WO2005019451A1

WO2005019451A1 - Nucleic acid construct for expressing pdk-1 activity inhibitor

Info

Publication number: WO2005019451A1
Application number: PCT/JP2004/004536
Authority: WO
Inventors: Norio Sakai
Original assignee: Japan Science And Technology Agency
Priority date: 2003-08-22
Filing date: 2004-03-30
Publication date: 2005-03-03
Also published as: JPWO2005019451A1

Abstract

It is intended to provide a means of regulating the phosphorylation activity of PDK-1 and a means of treating diseases caused by disorder in this activity. A nucleic acid construct for expressing an inhibitor of 3-phosphoinositide-dependent protein kinase 1 activity which contains a nucleic acid encoding a substance capable of inhibiting the activity of the phosphoinositide-dependent protein kinase 1 in vivo; a support for the expression of an inhibitor of 3-phosphoinositide-dependent protein kinase 1 activity in a mammalian cell which carries the nucleic acid construct for the expression as described above; a remedy and a therapeutic method for diseases caused by disorder in the phosphorylation activity of 3-phosphoinositide-dependent protein kinase 1 which contains the nucleic acid construct for the expression as described above or the support for the expression as described above as the active ingredient; and the nucleic acid construct for the expression as described above or the support for the expression as described above aiming at the production of a medicinal composition for the above diseases.

Description

Specification

Nucleic acid construct for expression of PDK-1 activity inhibitor

The present invention relates to a substance that inhibits the activity of 3-phosphoinositide-dependent protein kinase PDK-1, which is a kinase. More specifically, the present invention relates to a nucleic acid construct for expression capable of expressing an activity inhibitory substance that suppresses protein phosphorylation by PDK-1 in a living body, a carrier for expressing the PDK-1 activity inhibitory substance, The present invention relates to a therapeutic agent for a disease caused by abnormal phosphorylation activity by PDK-11, a method for treating the disease, and the like. Background art

For the treatment of cancer, for example, an attempt has been made to kill cancer cells themselves using an anticancer drug or the like.

It is known that the anticancer agent causes apoptosis in various cells [Gunji, H. et al., Cancer Research (Cancer Res.), Vol. 51, 741-743. Pp. 1991; Kaufman, SH, Cancer Research, Vol. 49, pp. 5870-5878, 1989]. However, for example, a protein kinase inhibitor such as staurosporine may cause apoptosis non-specifically in addition to a target cancer cell, and may be difficult to use as an anticancer drug. There is. Disclosure of the invention

One aspect of the present invention is a 3-phosphoinositide-dependent protein kinase. Expression of an activity inhibitor that can inhibit phosphorylation activity, expression of the activity inhibitor in vivo, and treatment of diseases caused by abnormal phosphorylation activity of 3-phosphoinositide-dependent protein kinase. Inhibiting the activity of 3-phosphoinositide-dependent protein kinase, which makes it possible to express at least one of the activity-inhibiting substances and to mass-produce the activity-inhibiting substances in an individual at least. An object of the present invention is to provide a nucleic acid construct for expressing a substance. Another aspect of the present invention is to provide a stable supply of the activity inhibitor, to express the activity inhibitor with high efficiency in vivo, and to express the activity inhibitor with high efficiency in vivo. Another object of the present invention is to provide a carrier for expressing a substance inhibiting the activity of 3-phosphoinositide-dependent protein kinase, which enables at least one of the following: specifically expressing the activity inhibitor in a desired cell or the like. . Furthermore, another aspect of the present invention relates to a disease caused by abnormal activity of 3-phosphoinositide-dependent protein kinase, specifically, an individual suffering from cancer or the like, a desired cell, particularly a cancer cell. Expressing an inhibitor of 3-phosphoinositide-dependent protein kinase activity to inhibit the phosphorylation activity of the 3-phosphoinositide-dependent protein kinase; causing apoptosis in desired cells, particularly cancer cells It is to provide a therapeutic agent that enables at least one of the above. Yet another aspect of the present invention is to provide a method for treating a disease caused by an abnormality in the activity of 3-phosphoinositide-dependent protein kinase. Furthermore, still another aspect of the present invention relates to a 3-phosphoinositide-dependent protein kinase activity inhibitor for the manufacture of a pharmaceutical composition for treating a disease caused by abnormal 3-phosphoinositide-dependent protein kinase activity. The present invention relates to the use of a nucleic acid construct for expression or a carrier for expression. The gist of the present invention is:

[1] (A) the amino acid sequence represented by SEQ ID NO: 1 or 2, (B) an amino acid sequence having at least one amino acid residue substitution, deletion, insertion or addition in the amino acid sequence shown in SEQ ID NO: 1 or 2, and

(C) A sequence calculated by aligning the amino acid sequence represented by SEQ ID NO: 1 or 2 with the BLAST algorithm (Costo openg ap 11, Costoextendgap 1, expectvalue 10, wordsize 3) An amino acid sequence having at least 26% identity,

An amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO: 3,

(b) an amino acid sequence having a substitution, deletion, insertion or addition of at least one amino acid residue in the amino acid sequence represented by SEQ ID NO: 3, and

(c) Sequence identity calculated and aligned with the amino acid sequence shown in SEQ ID NO: 3 using the BLAST algorithm (Costtooeng 11, Costtoextendap ap 1, expectvalue 10, wordsize 3) Is at least 26% amino acid sequence,

A 3-phosphoinositide-dependent nucleic acid comprising an amino acid sequence selected from the group consisting of: and a nucleic acid encoding an activity inhibitor capable of inhibiting the activity of phosphoinositide-dependent protein kinase 1 in vivo. Nucleic acid construct for expression of an inhibitor of the activity of soluble protein kinase 1,

[2] the nucleic acid encoding the activity inhibitor is

(i) the amino acid sequence represented by SEQ ID NO: 4,

(ii) an amino acid sequence having at least one amino acid residue substitution, deletion, insertion or addition in the amino acid sequence represented by SEQ ID NO: 4, and

(iii) The amino acid sequence shown in SEQ ID NO: 4 was compared with the BLAST algorithm (Costtoopenga 11, Costostextend). an amino acid sequence whose sequence identity is at least 26%, which is aligned by gap1, expectvalue10, wordsize3),

Amino acid sequence selected from the group consisting of

The nucleic acid construct for expression according to the above (1), which is operably linked downstream of the nucleic acid encoding

(3) a nucleic acid encoding an activity inhibitor, expressed under the control of the telomerase promoter, the nucleic acid construct for expression according to (1) or (2),

[4] a carrier for expressing a 3-phosphoinositide-dependent protein kinase 1 activity inhibitory substance in a mammalian cell, which holds the expression nucleic acid construct according to any one of [1] to [3]. ,

(5) the expression nucleic acid construct or the nucleic acid construct according to any one of (1) to (3);

(4) a therapeutic agent for a disease caused by abnormal phosphorylation activity by 3-phosphoinositide-dependent protein kinase 1, comprising the expression carrier as an active ingredient,

(6) the therapeutic agent according to (5), wherein the disease is cancer or a viral infection;

(7) Abnormal phosphorylation activity of 3-phosphoinositide-dependent protein kinase 1, which comprises an expression product of the nucleic acid construct for expression according to any one of (1) to (3) above as an active ingredient. Remedy for the resulting disease,

[8] The expression product is

(I) an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7,

(II) an amino acid sequence having a substitution, deletion, insertion or addition of at least one amino acid residue in the sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7, and

(III) The amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7 is aligned by the BL AST algorithm (Costtoopengap11, Costtoetendgap1, expectvalue10, wordsize3), If the calculated sequence identity is at least An amino acid sequence that is also 26%,

The therapeutic agent according to (7), wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:

(9) The expression nucleic acid construct or the nucleic acid construct according to any one of (1) to (3).

[4] administering a therapeutically effective amount of the expression carrier described in the above to an individual suffering from a disease caused by abnormal phosphorylation activity of 3-phosphoinositide-dependent protein kinase 1, Method for treating diseases caused by abnormal phosphorylation activity by 1 and

[10] The above-mentioned [1]-, for producing a pharmaceutical composition for treating a disease caused by an abnormality in phosphorylation activity by 3-phosphoinositide-dependent protein kinase 1;

(3) Use of the nucleic acid construct for expression according to any one of the above or the carrier for expression according to the above (4),

About. Brief Description of Drawings

FIG. 1 shows a schematic diagram of the primary structure of a PDK-1 activity inhibitor bound to green fluorescent protein (GFP). Panel A shows PKBAL-PIF-GFP, Panel B shows Lynsig-PKBAL-PIF-GFP, Panel C shows δAL-PIF-GFP, Panel D shows Lyn sig-SAL — PIF—Indicates GFP.

Fig. 2 shows the distribution of expression of AL-PIF-GFP and Lynsig- (5AL-PIF-GFP in cells. Panel A shows the distribution of expression of 5AL-PIF-GFP, and Panel B shows the distribution of expression of 5AL-PIF-GFP. 2 shows the expression distribution of Lyn sig—SAL—PIF—GFP In the figure, the scale is 10 m.

FIG. 3 shows the morphological change of the cells due to 3AL-PIF-GFP in the cells. Panel A shows the fluorescence image of GFP, and Panel B shows the results of nuclear staining. Show. In the figure, the scale bar indicates 10 m.

FIG. 4 shows the results of examining the effect of δ ALP IF-GFP on phosphorylation of the activation loop of PKB. In the figure, Panel A shows the results of Western blot analysis using an anti-GFP antibody, and Panel B shows the results of Western blot analysis using an anti-phosphorylated PKB (Thr308) antibody. In each panel, lane 1 shows the case where a protein sample (20 / g) prepared from COS-7 cells in which PKB cn-GFP and GFP were co-expressed was used. This figure shows the case of using a protein sample (20 g) prepared from COS-7 cells in which GFP and 6ALP IF-GFII were co-expressed. BEST MODE FOR CARRYING OUT THE INVENTION

The invention provides, in one aspect,

(A) the amino acid sequence of SEQ ID NO: 1 or 2,

(B) an amino acid sequence having at least one amino acid residue substitution, deletion, insertion or addition in the amino acid sequence shown in SEQ ID NO: 1 or 2, and

(C) Alignment of the amino acid sequence shown in SEQ ID NO: 1 or 2 with the BLAST algorithm (Costtooeng ap 11, Costtoextendgap 1, expectvalue 10, wordsize 3), and the calculated sequence is the same. An amino acid sequence having at least 26%

An amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO: 3,

(c) The BLAST algorithm (Co sttoopeng ap 11, Co sttoextend amino acid sequences that are aligned according to g ap1, expectvalue10, wordsize3) and have a calculated sequence identity of at least 26%,

3-phosphoinositide-dependent protein kinase 1 comprising an amino acid sequence selected from the group consisting of: and a nucleic acid encoding an activity inhibitor capable of inhibiting the activity of PDK-11 in vivo. (Hereinafter referred to as “PDK-1”).

Since the nucleic acid construct for expression of the present invention contains a nucleic acid encoding any one of the amino acid sequences (A) to (C) and any one of the amino acid sequences (a) to (c), PDK-1 activity inhibitor can be expressed. Therefore, by supplying such a nucleic acid construct for expression to an affected part of a disease caused by abnormal PDK-1 activity, the abnormal PDK-1 activity can be corrected. In addition, in individuals and cells requiring treatment for diseases caused by abnormal phosphorylation activity by PDK-1, an activity inhibitor that can inhibit the phosphorylation activity of PDK-1 is expressed, It has an excellent effect that it can be easily manufactured.

As used herein, the term “disease caused by abnormal PDK-1 phosphorylation activity” means a disease caused by abnormal PDK-1 activity, for example, cancer, virus infection and the like. No. Specific examples of the cancer include cancers in which cell proliferation is promoted based on an increase in the activity of PKB activated by PDK-1. As used herein, “abnormal phosphorylation activity due to PDK-1” refers to PDK-1 when compared to healthy individuals, unaffected individuals, individuals in an unaffected state, etc. It means a state deviating from the average value of phosphorylation activity.

In addition, in the present specification, “a PDK-1 activity inhibitor” refers to a substance that is phosphorylated by PDK-1.

“Inhibition of PDK-1 activity” refers to, for example, a protein phosphorylated by PDK-1. It can be evaluated by using an antibody that specifically recognizes the amino acid residue and confirming that the amount of the PDK-1 substrate protein recognized by this antibody is reduced.

The affinity of the PDK-1 activity inhibitor should be such that it suppresses the activation of PKB by PDK-1 in vivo and sufficiently exerts the activity of inducing apoptosis in cells.

Phosphorylation of the activity inhibitor by PDK-1 is carried out, for example, by using an antibody that specifically recognizes an amino acid residue phosphorylated by PDK-1 (mainly a threonine residue or the like). Whether or not a substance is recognized by this antibody can be measured by the immunoblot method or the like.

In addition, the affinity between PDK-1 and an activity inhibitor can also be evaluated by its binding activity. The binding activity is not particularly limited, but can be measured by a two-hybrid method, co-immunoprecipitation, surface plasmon resonance measurement, or the like.

Specifically, in the case of the two-hybrid method,

(1) integrating a nucleic acid encoding an activity inhibitor into a p ree vector to obtain a pr e plasmid;

(2) a step of introducing the obtained pREy plasmid into yeast CEGY48 [p8oplacZ] to obtain a clone retaining the pREy plasmid;

(3) integrating the PDK-1 gene into a bait vector to obtain a bait plasmid;

(4) introducing the obtained b ait plasmid into the pREy plasmid-carrying clone obtained in step (2) to obtain a clone;

(5) The clone obtained in (4) above is cultured on a leucine-deficient plate for 3 days, and the interaction with the activity inhibitor in yeast cells is determined by the ability to form a colony on the leucine-deficient plate. Evaluating by:

The formation of a colony on a leucine-deficient plate can be evaluated as an indicator of the binding between PDK-1 and the activity inhibitor. In the case of co-immunoprecipitation,

(1) obtaining a PDK-1-FLAG expression plasmid expressing PDK-1 having a FLAG sequence downstream of the signal sequence;

(2) obtaining an HA-activity inhibitor expression plasmid capable of expressing the activity inhibitor having an HA sequence on the N-terminal side;

(3) Transfect the cells with the PDK-1-FLAG expression plasmid obtained in step (1) and the HA-activity inhibitor substance expression plasmid obtained in step (2). Obtaining transfectants;

(4) culturing the cotransfectant obtained in the step (3) to obtain cultured cells;

(5) a step of obtaining a cell extract from the cultured cells obtained in the step (4);

(6) co-immunoprecipitation by adding an anti-FLAG antibody or an anti-HA antibody to the cell extract obtained in step (5);

(7) detecting the co-immunoprecipitation by Western blot analysis; and performing PDK-1 to detect that the precipitation of the complex is detected using either the anti-FLAG antibody or the anti-HA antibody. It can be evaluated as an index of binding to the activity inhibitor.

In the case of surface plasmon resonance analysis, a solution containing an activity-inhibiting substance is sent at a constant flow rate to a chip on which PDK-1 is immobilized, and optical (fluorescence, fluorescence polarization, etc.) detection means, mass spectrometry The PDK-1 and the activity inhibitor are detected by detecting means such as a matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer (MALD I-TOF MS, electrospray ionization mass spectrometer: ES I-MS). When a sensorgram showing the formation of a complex consisting of the following is presented, it can be evaluated as an indicator of the binding between the PDK-1 and the activity inhibitor. According to such surface plasmon resonance analysis, the reaction rate and the like can also be evaluated. “In vivo, the activity of inhibiting PDK-1 activation of PKB and inducing apoptosis in cells” (also called apoptosis-inducing activity) refers to chromatin condensation, fragmentation of cell nuclei, and formation of apoptotic bodies. And the like, measurement of an increase in the activity of an enzyme (such as lactate dehydrogenase) leaking from cells upon cell death, staining with trypan blue, etc., and evaluation using the TUNEL method.

In the above (A), the amino acid sequence represented by SEQ ID NO: 1 is the sequence of activate 1 oop of PKBZAkt, and the amino acid sequence represented by SEQ ID NO: 2 is the amino acid sequence of δPKC. Since the activity inhibitor obtained by using the nucleic acid construct for expression of the present invention has the amino acid sequence shown in SEQ ID NO: 1 or 2, it can activate DK by PDK-1 in vivo. It exhibits the surprising property of suppressing it.

In the present invention, the activation on loo exhibits substantially the same function as that of the activation on loo, ie, exhibits affinity for PDK-1, apoptosis-inducing activity, and the like, and is phosphorylated by PDK-1; If it acts as a pseudosubstrate and suppresses the phosphorylation function of PDK-1, a derivative sequence of the amino acid sequence represented by SEQ ID NO: 1 or 2 [for example, the amino acid sequence of (B) or (C) above] ].

In the amino acid sequence of (B), the number of substitutions, deletions, insertions or additions of amino acid residues is substantially the same as that of the actiVation loop, that is, the affinity with PDK-1. It can be appropriately set within a range that shows apoptosis-inducing activity and the like and is phosphorylated by PDK-1.

In the present specification, “at least one” means one or more, preferably one or several.

In the amino acid sequence of the above (C), the conditions in the algorithm used for calculating the sequence identity include, for example, Costo openg ap 11, Costoextend g ap 1, epectvalue 10, w ordsize 3. Other values may be the commonly used default values.

The sequence identity between the amino acid sequence of (C) and the amino acid sequence shown in SEQ ID NO: 1 or 2 has a function substantially equivalent to that of the above-mentioned activation on loop, ie, an affinity with PDK-1. It should be within the range that shows the activity, apoptosis-inducing activity, etc., and is phosphorylated by PDK-1. Under the above-mentioned conditions, when appropriately calculated by the BLAST algorithm and calculated, at least 26% is preferable. Is 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and still more preferably 95% or more. It is desirable.

In the amino acid sequences (B) and (C), the amino acid sequence (Thr Phe Cys Gly Thr) shown in SEQ ID NO: 9 is desirably conserved. Further, the activity inhibitor obtained by using the nucleic acid construct for expression of the present invention comprises, in addition to the amino acid sequence selected from the group consisting of (A) to (C), the following (a) to (c): Since it has an amino acid sequence selected from the group, it has an excellent effect of suppressing PDK-1 activation of PKB-1 in vivo more efficiently and inducing apoptosis in cells.

In the amino acid sequence of the above (a), the amino acid sequence shown in SEQ ID NO: 3 is the sequence of a PDK-1 interacting fragment (PIF).

The amino acid sequence represented by SEQ ID NO: 3 binds a polypeptide having the amino acid sequence to protein kinase C-related kinase-2 (PRK2), which is one of AGC kinases which is an original substrate of PDK-1. A derivative sequence of the amino acid sequence represented by SEQ ID NO: 3 [for example, the amino acid sequence of (b) or (c) above] may be used as long as it exhibits the activity of

In the amino acid sequence of (b), the number of substitutions, deletions, insertions or additions of amino acid residues is substantially the same as that of PIF, that is, the affinity with PDK-1. Etc. can be appropriately set within a range indicating the above.

In the amino acid sequence (c), the sequence identity may be within a range that exhibits substantially the same function as the PIF, that is, the affinity with PDK-1.

Further, the nucleic acid construct for expression of the present invention,

(i) the amino acid sequence represented by SEQ ID NO: 4,

(iii) The amino acid sequence represented by SEQ ID NO: 4 is aligned and calculated using the BLAST algorithm (Cost opopenp ap 11, Co sttoextend ap 1, Expectvalue 10, femalesize 3). An amino acid sequence having a sequence identity of at least 26%,

Amino acid sequence selected from the group consisting of

May further be included. Examples of such a nucleic acid construct for expression include a construct in which a nucleic acid encoding an activity inhibitor is operably linked downstream of an amino acid sequence selected from the group consisting of (i) to (iii). Said

According to the expression nucleic acid construct containing the amino acid sequence selected from the group consisting of (i) to (iii), the activity inhibitor can be expressed in the cell membrane.

In the amino acid sequence of the above (i), the amino acid sequence shown in SEQ ID NO: 4 is the N-terminal sequence of the lip mouth synthase Lyn present in the cell membrane, and the amino acid sequence shown in SEQ ID NO: 4 By palmitating the portion consisting of, the polypeptide having such an amino acid sequence is localized on the cell membrane.

Therefore, in the present invention, a derivative sequence of the amino acid sequence of SEQ ID NO: 4 [the amino acid sequence of (ii) or (iii)] as long as it is palmitized and exhibits the ability to localize to the cell membrane It may be.

In the amino acid sequence (ii), substitution, deletion, insertion or Like the N-terminal sequence of Lyn, the number of additions can be appropriately set within a range that exhibits localization ability to cell membranes by palmitization.

In the amino acid sequence of (iiii), the sequence identity may be in a range that, as in the case of the N-terminal sequence of Lyn, exhibits localization ability to a cell membrane by being palmitized.

The palmitination can be evaluated by fractionating cells into a cell membrane component and a cytoplasmic component, and detecting that a large amount of a PDK-1 activity inhibitor is expressed in the cell membrane component by an immunoblot method. In addition, the ability to localize to the cell membrane can be confirmed by fluorescence microscopy. Whether the Lyn-added PDK-1 activity inhibitor fused with GFP is Lyn-added or more localized to the cell membrane than the PDK-1 substance It can be evaluated by observing at.

The nucleic acid encoding the activity inhibitor used in the nucleic acid construct for expression of the present invention is obtained by synthesizing the corresponding nucleic acid based on the amino acid sequence described in the sequence listing, and converting the nucleic acid encoding PKB or PKC delta into a type III nucleic acid. And can be obtained by performing PCR.

Further, in the expression nucleic acid construct of the present invention, the expression of the activity inhibitor can be specifically performed in cancer cells by expressing the nucleic acid encoding the activity inhibitor under the control of a promoter such as a telomerase promoter. Can be. Specifically, for example,

1. a construct in which a nucleic acid encoding an activity inhibitor is operably linked downstream such as telomerase promoter

2. A construct in which a nucleic acid encoding a tetracycline transactivator is operably linked downstream of a promoter such as a telomere-zepromoter, and a nucleic acid encoding an activity inhibitor, wherein the nucleic acid encodes the transactivator. Combination with an operably linked construct downstream of a promoter that is activated by an activator

Etc. As used herein, the term "operably linked" refers to a state in which polypeptides exhibiting a desired function are linked so as to be normally expressed. For example, "a nucleic acid is operably linked downstream of a promoter" means that an expression product is produced from the nucleic acid under the control of the promoter.

Examples of the transactivator and the promoter activated by the transactivator include a tetracycline transactivator and a Tet Op promoter. When the tetracycline transactivator and the Tet Op promoter are used, the expression of the activity inhibitor by the nucleic acid construct for expression can be stopped, so that the time-specific expression control of the activity inhibitor, for example, moderate It is suitable for the case where the expression is stopped so as to act on the target, or the case where the expression is performed only for a certain period of time.

The activity inhibitor expressed by the expression nucleic acid construct is not particularly limited,

(I) an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7,

(III) The amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7 is aligned and calculated by the BL AST algorithm (Costtoopengap 11, Costtoextendaga 1, expectvalue 10, female size 3). An amino acid sequence having a sequence identity of at least 26%,

And a polypeptide containing an amino acid sequence selected from the group consisting of: The nucleic acid construct for expression of the present invention can stably express an activity-inhibiting substance by being held in a carrier suitable for introducing a nucleic acid into cells or the like. Therefore, the nucleic acid construct for expression of the present invention exhibits an excellent effect that an activity inhibitor can be stably supplied. According to the nucleic acid construct for expression of the present invention, The activity inhibitor can be more efficiently introduced into an individual, a cell, or the like, and the activity inhibitor can be expressed with high efficiency in an individual, a cell, or the like, and the activity inhibitor can be specifically expressed in a desired cell or the like. It has an excellent effect of being able to do it.

Therefore, another aspect of the present invention is a carrier for expressing a PDK-1 activity inhibitor in mammalian cells, which holds the expression nucleic acid construct.

Suitable carriers for introducing nucleic acids into cells and the like include mammals such as viral vectors, for example, adenovirus vectors, herpes virus vectors, adeno-associated virus vectors, Sindbis virus vectors, retrovirus vectors, etc. Examples include a vector for cells, a ribosome containing a plasmid vector, and gold particles having a plasmid vector attached thereto. Among them, from the viewpoint of expression efficiency in cells, a viral vector having a transient expression ability is preferable.

In addition, the expression nucleic acid construct and the expression carrier of the present invention can be used for treating a disease caused by abnormal PDK-1 phosphorylation activity.

Therefore, another aspect of the present invention is a therapeutic agent for a disease caused by abnormal phosphorylation activity by PDK_1.

One major feature of the therapeutic agent of the present invention is that it contains the expression nucleic acid construct or the expression carrier as an active ingredient. Since the therapeutic agent of the present invention contains the nucleic acid construct for expression or the carrier for expression, in an individual suffering from a disease caused by abnormal phosphorylation activity by PDK-1, the activity inhibitory substance can be expressed in a desired cell in an individual. It exerts an excellent effect of inhibiting the phosphorylation activity of PDK-1, suppressing the targeting mechanism by PDK-1, and causing apoptosis in desired cells.

Therefore, the therapeutic agent of the present invention is suitable for treating a disease caused by abnormal PDK-1 phosphorylation activity, more specifically, a disease in which apoptosis induction leads to a therapeutic effect.

Diseases caused by abnormal phosphorylation activity by PDK-1 are the same as those described above. Yes, for example, cancer, for example, cancer, in particular, cancer in which cell proliferation is promoted based on an increase in the activity of PKB activated by PDK-1, cancer, and viral infection. Examples of the cancer include, for example, breast cancer and the like, which can promote cell proliferation based on an increase in the activity of PKB activated by PDK-1.

The therapeutic agent of the present invention can be administered by an administration route according to a target cell, a target tissue, a target organ, and the like. Examples of the administration route include intravenous, arterial, subcutaneous, intradermal, intramuscular, and topical.

The content of the nucleic acid construct for expression or the carrier for expression in the therapeutic agent of the present invention can be appropriately set according to the disease, the age and weight of the patient, etc., but the activity inhibitor is expressed in vivo. Then, an amount sufficient to suppress the targeting mechanism by PDK-1 more efficiently and induce apoptosis in cells (a therapeutically effective amount) may be sufficient.

The therapeutic agent of the present invention includes, in addition to the nucleic acid construct for expression or the carrier for expression, a pharmacologically acceptable auxiliary agent, for example, a buffer, an excipient, a binder, and a stable agent capable of stably holding a nucleic acid. Agents, solubilizing agents, isotonic agents and the like.

The administration method of the therapeutic agent of the present invention includes monocalcium phosphate coprecipitation method, direct injection method using a microscopic glass tube, gene transfer method using ribosome, method of transferring into cells by a particle gun, and method of transfer using a positively charged polymer. And the like.

For the therapeutic agent of the present invention, for example, in the case of cancer, pharmacological evaluation can be performed by the following animal experiment. An appropriate dose of a therapeutic agent is administered to a nude mouse cancer model in which tumor formation has been confirmed in an appropriate number of doses, and at the same time, the change in tumor diameter is observed. When the tumor group regression was observed in the administration group compared to the control group in which the treatment group was not administered to the mice to which the therapeutic agent was administered or the mice treated with other treatments, the animals were treated as animals. It serves as an indicator that the cancer was successfully treated at the experimental level. Apoptosis may be detected in tissues, cells, and the like. In addition, human clinical trials with the therapeutic agents of the present invention include, for example, The size of the tumor is measured by periodic observation (photographing, etc.), recorded by CT scan, MRI, etc., and the estimated tumor volume is calculated from the orthogonal major axis and minor axis to calculate tumor growth. The therapeutic effect can be evaluated based on the evaluation index corresponding to the type of tumor. The presence or absence of apoptosis may be evaluated by morphological observation of tissues, cells, and the like.

Further, in still another aspect, the present invention provides a method for treating the expression nucleic acid construct or the expression carrier for an individual suffering from a disease caused by abnormal phosphorylation activity of 3-phosphoinositide-dependent protein kinase 1. A method for treating a disease caused by abnormal phosphorylation activity of 3-phosphoinositide-dependent protein kinase 1, characterized by administering an upper effective dose.

The treatment method of the present invention is performed in the same manner as in the case where the therapeutic agent is administered to an individual. In addition, the evaluation of the therapeutic effect by such a treatment method can be performed in the same manner as the pharmacological evaluation and the clinical test of the therapeutic agent.

Further, according to the present invention, expression of a substance inhibiting the activity of 3-phosphoinositide-dependent protein kinase for the production of a pharmaceutical composition for treating a disease caused by abnormal activity of 3-phosphoinositide-dependent protein kinase. Also provided is the use of a nucleic acid construct for expression or a carrier for expression. According to the nucleic acid construct for expression or the carrier for expression of the present invention, it becomes possible to develop a method for treating a disease caused by abnormal activity of 31-phosphoinositide-dependent protein kinase. Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples. Example 1

A portion corresponding to the activation loop of 3-phosphoinositide-dependent protein kinase (referred to as “PDK-1”; SEQ ID NO: 1) and a PDK-1 interacting fragment (referred to as “PIF”; SEQ ID NO: 1) No .: 3) A nucleic acid construct (PKBAL-PIF) having two copies of the nucleic acid encoding was prepared. Further, a nucleic acid encoding GFP is arranged downstream of the nucleic acid construct (PKBAL-PIF) so that green fluorescent protein (GFP) is arranged on the C-terminal side of the expression product of the nucleic acid construct (PKBAL-PIF). (PKBAL-PIF-GFP) was obtained.

Then, the PKBAL-PIF was integrated into the mammalian cell expression vector pTB701 at a position downstream of the SV40 promoter to obtain a recombinant vector for PKBAL-PIF expression. In addition, the PKBAL-PIF-GFP was incorporated downstream of the SV40 promoter of the mammalian cell expression vector pTB701 to obtain a recombinant vector for PKBAL-PIF-GFP expression.

Further, a nucleic acid encoding a peptide consisting of the amino acid sequence (SEQ ID NO: 4) of the N-terminal region of tyrosine kinase Lyn present in the cell membrane is used as a nucleic acid construct, and the expression product of the nucleic acid construct (PKBAL-PIF) and The nucleic acid construct (Lynsig-PKBAL-PIF) and the nucleic acid construct (LKB-PKBAL-PIF) were linked to each nucleic acid construct so that the nucleic acid construct (PKBAL-PIF-GFP) expression product was added to each N-terminal side. Each of the structures (Lyn sig-PKBAL-PIF-GFP) was obtained.

In addition, a nucleic acid construct having two copies of a nucleic acid encoding a portion corresponding to an activation loop of PKC and a PIF of δ PKC, <5 which is a substrate of PDK-1 like PKB / Akt. (0AL-PIF). Further, a nucleic acid encoding GFP is arranged downstream of the nucleic acid construct ((5AL-PIF) so that GFP is arranged on the C-terminal side of the expression product thereof, and the nucleic acid construct (< 5 AL—PIF—GFP) was obtained.

Then, the SAL-PIF was incorporated downstream of the SV40 promoter of the mammalian cell expression vector pTB701 to obtain a recombinant vector for expression of δAL-PIF. Further, the <5AL-PIF-GFP was incorporated into the mammalian cell expression vector ρTB701 downstream of the SV40 promoter. A recombinant vector for expression was obtained.

Furthermore, a nucleic acid encoding a peptide consisting of the amino acid sequence (SEQ ID NO: 4) of the N-terminal region of the tyrosine kinase Lyn present in the cell membrane is obtained by using the nucleic acid construct (SAL-PIF) Each of the nucleic acid constructs (Lyn sig-5AL-PIF) and the nucleic acid construct (Lyn sig-5AL-PIF) are linked to each nucleic acid construct so that the nucleic acid construct (SAL-PIF-GFP) is added to the N-terminal of each expression product. sig- (5AL-PIF-GFP) was obtained.

Each of the obtained recombinant vectors was introduced into mammalian cell line COS-7 (supplied by RIKEN Cell Punk) by the electoporation method.

FIG. 1 shows a schematic diagram of the primary structure of the GFP-binding expression product among the obtained expression products.

In addition, a recombinant adenovirus was prepared by the homologous recombination method using each of the nucleic acid constructs. For the production of the recombinant adenovirus, Stratagene, trade name: pAdEASY System, trade name: AdEasy Adenovira1VectorSistem was used.

HEK 293 cells (Haccho (: 〇 CRL-1573), containing 100 units / m1 penicillin G sodium, 100 gZm1 streptomycin and 10% by weight of fetal serum ο; — cells on a 60 mm culture plate in MEM The cells were inoculated to a number of 2 to 5 × 10 ⁵ (about 100 cells / mm ² ) and cultured at 37 ° C. in a humidified gas phase containing 5% by volume of CO ₂ .

After 12 to 24 hours, HEK293 cells were transfected with the obtained recombinant adenovirus vector. After 4 to 7 days, the cells detached from the plate were centrifuged at 1500 × g for 5 minutes to obtain a virus supernatant. The virus titer value of the obtained recombinant adenovirus supernatant was measured. As a result, an adenovirus vector expressing the nucleic acid construct was obtained. Example 2

Of the expression vectors obtained in Example 1 above, each of a PIF-GFP expression plasmid vector and a Lysig-a AL-PIF-GFP expression plasmid vector was subjected to the monkey kidney method by electoporation. Cancer cell C

Introduced in OS-7.

Twenty-four hours after the introduction, each of the δAL-PIF-GFP-expressing cells and the Lyssig-δAL-PIF-GFP-expressing cells was observed using GFP fluorescence as an index. The result is shown in figure 2.

As a result, as shown in Fig. 2, <5 AL-PIF-GFP expression products were mainly expressed in the cytoplasm, and Lyn sig-δAL-PIF-GFP expression products were mainly expressed in the cell membrane. Expression was seen.

Forty-eight hours after the introduction, (5AL-PIF-GFP-expressing cells and Lynxig-ΔAL-PIF-GFP-expressing cells were each observed using GFP fluorescence as an index.

As a result, both the cells expressing SAL-PIF-GFP and the cells expressing Lynsig- <5AL-PIF-GFP caused spherical morphological changes. In addition, each cell was stained with Hoechst 33342, and as a result, all cells showing morphological changes showed chromatin aggregation and nuclear fragmentation, indicating that apoptosis was occurring. . In addition, FIG. 3 shows the result in the case of (5AL-PIF-GFP-expressing cells) as a representative example.

Therefore, it was found that the expression product of the nucleic acid construct expressed by the expression vector obtained in Example 1 inhibits the activity of 3-phosphoinositide-dependent protein kinase 1 and induces apoptosis. Example 3

Tetracycline transactivation under the control of the telomerase promoter Then, a vector 1 for expressing the tetracycline transactivator 1 in a cancer cell-specific manner is prepared.

In addition, a Tet Op minimal promoter is ligated upstream of the nucleic acid construct of Example 1, and an expression vector is prepared in the same manner as in Example 1 (Vector 2).

Each of the cancer cells established with the obtained Vector 1 and Vector 2 and non-established cancer cells is introduced.

As a result, the effect of the expression product of the nucleic acid construct is evaluated based on the fact that the cell undergoes a morphological change in a spherical shape and exhibits chromatin aggregation and nuclear fragmentation. Example 4

Vector 11 and Vector 2 obtained in Example 3 are intravascularly administered to a solid cancer model or locally injected into cancer cells of the model.

As a result, the effect of the expression product on cancer is evaluated using the change in the size of solid cancer as an index. Example 5

In COS-7 cells, PKBa-GFP was co-expressed with <5 ALPIF-GFP or GFP as a control.

Next, Western plot analysis was carried out using an anti-GFP antibody or an anti-phosphorylated PKB (Thr308) antibody that recognizes phosphorylated threonine of activatoloop of PKB. Fig. 4 shows the results.

As shown in FIG. 4, panel A, it can be seen that each of the expressed target proteins has the target size. Also, the expression level of PKB-GFP is? 8? 0? And 0 ?? were co-expressed (: 03-7 cells and? 1 ^ 80! -P and COS-7 cells co-expressed with δ ALP IF-GFP. Also, as shown in Panel B, the band of PKB «-GFP was thinner when <5 ALP IF-GFP was co-expressed (lane 2) than when GFP was co-expressed (lane 1). It is recognized that <5 ALP IF-GFP suppresses the phosphorylation of the PKB CK-GFP activation loupe. This antibody also recognizes phosphorylation of δ ALP IF-GFP. Example 6

Among the expression vectors obtained in Example 1, each of the pseudo-substrate δ AL-PIF-GFP expression plasmid vector and the pseudo-substrate Lyn sig——PIF—GFP expression plasmid vector was subjected to the electoral port method. As a result, the cells were introduced into cancer-derived cultured cells (COS-7 cells and PC-12 cells).

The expression vector of the pseudosubstrate and the expression plasmid vector of the fusion protein of GST and PDK-1 (GST-PDK-1) were transformed into a cancer-derived cultured cell (COS-7 cell) by electoporation. And PC-12 cells).

The expression plasmid vector for GST-PDK-1 is obtained by adding a cDNA encoding daryuthion-1S-transferase (GST) to the 5 'side of a cDNA encoding human PDK-1. This is a vector prepared by ligating the obtained nucleic acid construct to the same plasmid vector as in Example 1 above. As a control, an expression vector for GFP was introduced into each of the C〇S-7 cells and PC-12 cells instead of the pseudosubstrate expression vector.

48 hours after gene transfer, apoptotic cells were counted among cells exhibiting GFP-derived fluorescence, and the apoptosis induction rate was calculated. In addition, apoptosis-induced cells were stained by a conventional method using cell nuclei, and determined under a fluorescence microscope using chromatin aggregation and nuclear fragmentation as indices.

Table 1 shows the results for COS-7 cells, and Table 2 shows the results for PC-12 cells. You. In the table, * indicates p <0.05 Vs GFP [Bon ferr on imultiplec omp aris on stest], # indicates p 0.05 and only pseudo substrate, and ## indicates p 0.01. V s pseudo substrate only [unp airedt-test] is shown. The results show the average of triplicates. table 1

As a result, as shown in Tables 1 and 2, the expression of δAL-PIF-GFP was higher in both COS-7 cells and PC-12 cells when compared to the control cells expressing only GFΡ. As a result, the percentage of cells showing apoptosis significantly increased.

Also, as shown in Tables 1 and 2, when Lyn sig-δAL-PIF-GFP was expressed, compared to cells expressing only GFP as a control, COS-7 cells had However, it caused a significant increase in the proportion of cells showing apoptosis, and the number of apoptosis-inducing cells also increased in PC-12 cells.

Therefore, it was found that expression of the pseudosubstrate δAL-PIF-GFP and the expression of the pseudosubstrate Lyn sig-δAL-PIF-GFP significantly induced apoptosis in cultured cells derived from cancer. Furthermore, when GST-PDK-1 was expressed together with the pseudo substrate, as shown in Tables 1 and 2, co-expression of GST-PDK-1 and SAL-PIF-GFP caused apoptosis. Was significantly suppressed in both COS-7 cells and PC-12 cells.

These results suggest that exogenous overexpression of PDK-1 may reduce the effect of the pseudosubstrate, and that apoptosis by the pseudosubstrate occurs through endogenous PDK-1. Was suggested. Sequence listing free text

SEQ ID NO: 1 is a sequence of activatatiolop of PKB / Akt.

SEQ ID NO: 2 is a sequence of 6 PKC activatonioloop. SEQ ID NO: 3 is the sequence of a PDK-1 interacting fragment (PIF).

SEQ ID NO: 4 is a sequence of Lyn.

SEQ ID NO: 5 is a sequence of a PDK-1 inhibitor produced from PKBAL-PIF.

SEQ ID NO: 6 is a sequence of a PDK-1 GFP binding inhibitor produced from PKBAL-PIF-GFP.

SEQ ID NO: 7 is a sequence of a PDK-1 inhibitor produced from δAL-PIF.

SEQ ID NO: 8 is a sequence of a GFP binding inhibitor produced from δAL-PIF. SEQ ID NO: 9 is a partial sequence of activatatioop. Industrial applicability

According to the present invention, it is possible to develop therapeutic means for diseases, cancers, and the like caused by abnormal activity of 3-phosphoinositide-dependent protein kinase 1.

Claims

The scope of the claims

1. (A) the amino acid sequence of SEQ ID NO: 1 or 2,

(C) Sequence identity calculated by aligning the amino acid sequence shown in SEQ ID NO: 1 or 2 with the BLAST algorithm (Costtoopengap 11, Costtoextendgap 1, expectvalue 10, wordsize 3) Is at least 26% amino acid sequence,

An amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO: 3,

(c) Alignment of the amino acid sequence shown in SEQ ID NO: 3 with the BLAST algorithm (Costtooeng ap 11, Costtoextend gap 1, expectvalue 10, wordsize 3), and the calculated sequence is identical. An amino acid sequence having at least 26%

Comprising an amino acid sequence selected from the group consisting of: and a nucleic acid encoding an activity inhibitor capable of inhibiting the activity of phosphoinositide-dependent protein kinase 1 in vivo. A nucleic acid construct for expression of a phosphoinositide-dependent protein kinase 1 activity inhibitor.

2. The nucleic acid encoding the activity inhibitor is

(i) the amino acid sequence represented by SEQ ID NO: 4, (ii) an amino acid sequence having at least one amino acid residue substitution, deletion, insertion or addition in the amino acid sequence represented by SEQ ID NO: 4, and

(iii) The amino acid sequence represented by SEQ ID NO: 4 was aligned and calculated using the BLAST algorithm (Costtooeng ap 11, Costtoet nd g ap1, expectvalue 10, wordsize 3). An amino acid sequence having at least 26% sequence identity,

Amino acid sequence selected from the group consisting of

The nucleic acid construct for expression according to claim 1, which is operably linked downstream of a nucleic acid encoding

3. The nucleic acid construct for expression according to claim 1, wherein the nucleic acid encoding the activity inhibitor is expressed under the control of a telomerase promoter.

4. A carrier for expression of a substance that inhibits the activity of 3-phosphoinositide-dependent protein kinase 1 in mammalian cells, comprising the nucleic acid construct for expression according to any one of claims 1 to 3.

5. Due to the phosphorylation activity of 3_phosphoinositide-dependent protein kinase 1, comprising the expression nucleic acid construct according to any one of claims 1 to 3 or the expression carrier according to claim 4 as an active ingredient. Therapeutic agent for the disease.

6. The therapeutic agent according to claim 5, wherein the disease is cancer.

7. A 3-phosphoinositide-dependent protein kinase 1, comprising an expression product of the nucleic acid construct for expression according to any one of claims 1 to 3 as an effective component. For treating diseases caused by phosphorylation activity.

8. The expression product is

(I) an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7,

(III) The amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7 is aligned and calculated by the BLAST algorithm (Costtoopengap 11, Costtoextend gap 1, exectvalue 10, female size 3). An amino acid sequence having a sequence identity of at least 26%,

The therapeutic agent according to claim 7, which is a polypeptide containing an amino acid sequence selected from the group consisting of:

9. The nucleic acid construct for expression according to any one of claims 1 to 3 or the carrier for expression according to claim 4 is afflicted with a disease caused by abnormal phosphorylation activity of 3-phosphoinositide-dependent protein kinase 1. A method for treating a disease caused by abnormal phosphorylation activity of 3-phosphoinositide-dependent protein kinase 1, which is administered to a selected individual in a therapeutically effective amount.

10. The expression nucleic acid construct or claim according to any one of claims 1 to 3, for the manufacture of a pharmaceutical composition for treating a disease caused by abnormal phosphorylation activity by 3-phosphoinositide-dependent protein kinase 1. Item 14. Use of the expression carrier according to Item 4.