WO2006098463A1

WO2006098463A1 - Polynucleic acid-binding substance

Info

Publication number: WO2006098463A1
Application number: PCT/JP2006/305478
Authority: WO
Inventors: Masaya Ishibashi; Kazuo Harada; Hideki Nakashima
Original assignee: Shinka Soyaku Inc.
Priority date: 2005-03-14
Filing date: 2006-03-14
Publication date: 2006-09-21
Also published as: JPWO2006098463A1

Abstract

Disclosed is a substance which is bound to a polynucleic acid. This polynucleic acid-binding substance is characterized by containing a first portion having an electrophilic group and a second portion containing one or more substances selected from peptides, compounds and nucleic acids.

Description

Description Polynucleic acid binding substance Technical field

The present invention relates to a polynucleic acid binding substance. Specifically, the present invention includes a polynucleic acid binding substance comprising a first part having an electrophilic group and a second part having one or more selected from the group consisting of peptides, compounds and nucleic acids. About. Background art

Human immunodeficiency virus (HIV) is the causative virus for acquired immunodeficiency syndrome (AIDS). The virus has a transactivation region (TAR) and a Rev responsive element (RRE), which are found in unspliced or partially spliced HIV mRNA introns.

RNA encoding RRE and TAR interacts with specific HIV proteins during HIV replication. That is, when the HIV protein Rev recognizes RRE, it binds to RRE to form a Rev / RRE complex. The formation of the Rev / RRE complex is involved in the process of HIV protein Rev stimulating mRNA through the complex, and also involved in nuclear export signaling that is essential for Rev's Shuttling.

Thus, since the interaction of the Rev / RRE complex is essential when the virus grows, suppressing the function of this process greatly contributes to the suppression of the progression of acquired immune deficiency (AIDS). .

Treatment targeting Rev has been attempted using antisense and ribozymes. Furthermore, gene therapy using RRE decoy RNA is also being carried out.

The inventors have previously developed a method for screening RNA-binding proteins from cDNA libraries or peptide libraries, and have identified new substances (Japanese Patent Publication No. 11-511653, Hadas Peled-Zehavi, et al ., RNA 9, 252-261, 2003). The polypeptide obtained by these methods has a binding activity with a target RNA, and in particular has the ability to inhibit the proliferation of HIV by binding to the RRE of HIV (International Publication No. 1). 2005/007686 pamphlet). However, in order to more effectively use the polypeptide obtained for the treatment of HIV, it is desired to further improve its RNA binding activity and pharmacological effect. Disclosure of the invention

The present invention relates to a polynucleic acid-binding substance comprising a first part having an electrophilic group and a second part having at least one selected from the group consisting of peptides, compounds and nucleic acids, preferably an electrophilic group and a peptide. It is an object of the present invention to provide an RNA-binding peptide comprising Furthermore, an object of the present invention is to suppress the growth of HIV by increasing the polynucleic acid (preferably RNA) binding activity.

As a result of diligent research to solve the above problems, the present inventors have found that the addition of an electrophilic group to an RNA binding peptide increases the pharmacological action of the peptide, thereby completing the present invention. It came to.

That is, the present invention is as follows.

(1) A substance that binds to a polynucleic acid, comprising a first part having an electrophilic group and a second part having one or more selected from the group consisting of peptides and compound nucleic acids A polynucleic acid binding substance characterized by the above.

(2) The polynucleic acid-binding substance according to (1), wherein the polynucleic acid is NA.

(3) The polynucleic acid binding substance according to (2), wherein the RNA contains an RRE sequence site.

(4) The polynucleic acid-binding substance according to (1), wherein the second part contains a peptide.

(5) The peptide has the following formula I:

R-Q-R-X (I)

(X represents an amino acid residue other than R.)

(4) The polynucleic acid-binding substance according to (4), which is a peptide comprising the amino acid sequence represented by

(6) The peptide is the following peptide (a) or (b), a derivative thereof or a salt thereof: The polynucleic acid binding substance according to (4).

(a) Formula I:

R-Q-R-X (I)

(X represents an amino acid residue other than R.)

A peptide having an amino acid sequence of 12 to 23 residues including the amino acid sequence represented by

(b) Formula I:

R-Q-R-X (I)

(X represents an amino acid residue other than R.)

A peptide having an RNA binding activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in an amino acid sequence having a length of 12 to 23 residues including the amino acid sequence represented by

(7) The polynucleic acid-binding substance according to (5) or (6), wherein R contains at least 9 to 11 residues.

(8) The polynucleic acid-binding substance according to (4), wherein the peptide is the following peptide (a) or (b), a derivative thereof, or a salt thereof.

(a) a peptide comprising an amino acid sequence represented by SEQ ID NOs: 1 to 27

(b) A peptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NOs: 1 to 27, and having RNA binding activity

(9) The polynucleic acid binding substance according to (1), wherein the electrophilic group is a group represented by the following formula.

CH-C C——

Z I 0 I

(In the formula, Z represents a halogen atom.)

(10) The polynucleic acid binding substance according to (1), wherein a spacer is provided between the first part and the second part.

(1 1) A pharmaceutical composition comprising the polynucleic acid binding substance according to any one of (1) to (10).

(12) The pharmaceutical composition according to (11), which is an HIV growth inhibitor. (1 3) The pharmaceutical composition according to (1 1), which is an agent for preventing HIV infection.

(14) The pharmaceutical composition according to (11), which is a preventive or therapeutic agent for AIDS, the best mode for carrying out the invention

Hereinafter, the present invention will be described in detail.

Documents cited in this specification, as well as published publications, patent publications and other patent documents are incorporated herein by reference.

1. Summary of the present invention

The present invention relates to a polynucleic acid binding substance comprising a first part having an electrophilic group and a second part having one or more selected from the group consisting of peptides, compounds and nucleic acids. is there.

In the present specification, “polynucleic acid binding substance” means a substance capable of binding to a polynucleic acid. The polynucleic acid binding substance of the present invention is not limited by the strength of binding to polynucleic acid, as long as it has an activity of binding to polynucleic acid. In the present specification, the “polynucleic acid” means a nucleic acid connected in a chain form.

In this specification, “nucleic acid” means mRNA, rRNA, tRNA, viral RNA, decoy RNA, antisense RNA, ribozyme, siRNA, and other natural and artificial RNA (ribonucleic acid), cDNA, genome DNA, ssDNA (single strand DNA) ), DsDNA (double strand DNA) and other DNA (deoxyribonucleic acid), and PNA (peptide nucleic acid), preferably RNA. Examples of the virus include viruses such as HIV, FIV, SIV, BIV, and HTLV, which are retroviruses having a Rev responsive element (RRE) sequence site or a similar structure.

In the present invention, the second part preferably comprises a peptide.

2. Part 1

(1) Electrophilic group In the present invention, the first portion has an electrophilic group.

As used herein, “electrophilic group” means a reactive species (eg, cation, acid, oxidant) that lacks electrons. Electrophilic groups have the property of reacting in two ways: the ability to oxidize electrons from the other party and the formation of coordination bonds from the other party.

The electrophilic group included in the first part of the present invention may be any group as long as it functions as an electrophilic group. The electrophilic group contained in the first part of the present invention is, for example, the following formula:

(In the formula, Z means a halogen atom.)

(In the present invention, the group is also referred to as rZAc-j hereinafter).

In the present specification, the “halogen atom” is preferably a chlorine atom, a bromine atom, or an iodine atom, and more preferably a chlorine atom.

(2) Spacer

In the present invention, a spacer can be contained between the first part and the second part. By including a spacer, the reaction target can be a nucleophilic group present at an appropriate distance while maintaining the binding between the polynucleic acid and the second portion.

In the present invention, examples of the spacer include ether type aminoalkanoic acids such as GABA (y-aminobutanoic acid), ε-aminocaproic acid, or 5-amino-3-oxopentanoic acid. Also, a carboxylic acid having a plurality of amino groups branched can be used. These spacers can be bonded to the first part at the position of the amino group in the spacer, and can be bonded to the second part at the position of the carboxyl group in the spacer. it can.

In this case, it is preferable that the Ν terminal side of GABA is bonded to an electrophilic group (ClAc- in the following example) and the C terminal side is bonded to a second part (not shown) as follows.

When GABA is used as a spacer, the first part can be introduced by combining the GABA amino group and the chloroacetic acid carboxyl group.

(3) Connection with the second part

In the present invention, the first part and the second part are bound by an amide bond which is a covalent bond, and these can be reacted by a known method.

In the present invention, a plurality of first portions and / or spacers may be bonded to one second portion.

When the second part is a peptide, the first part or spacer can be attached to the N-terminal side and the no- or C-terminal side of the peptide.

When the carbonyl group is contained in the first part, the carbonyl group and the N terminus of the peptide or spacer are bonded by an amide bond. For example, when Z is a chlorine atom (ClAc-), the peptide or spacer to which the first moiety is added directly connects the peptide or spacer with the side-chain functional group protected to black mouth acetic acid. The ability to react with the condensing agent, and-after the conversion of chloroacetic acid to black acetic anhydride with the condensing agent, the reaction is carried out, or the reaction is carried out using an active ester such as black acetic acid para-diphenyl ester, or It can be obtained by reacting acetic chloride with a base. Further, for example, when the first part or spacer has a carbonyl group, the peptide can be synthesized sequentially following the carbonyl group of the first part or spacer.

Alternatively, when an amino group is contained in the first part, even when the amino group is bound to the C-terminus of the peptide or spacer, it is bound by an amide bond. The amide bond can be carried out by a person skilled in the art by a known method.

In addition, when the second part is a compound, the compound or spacer to which the first part is added is included in the compound or compound before addition when, for example, Z is a chlorine atom. The reaction force with a spacer to be converted to chloroacetic anhydride with a condensing agent or the reaction with an active ester such as chloroacetic acid paranitrophenyl ester or chloroacetic acid chloride Can be obtained by reacting with a base.

Further, when the second part is a nucleic acid, the electrophilic group or spacer and the nucleic acid can be bound by an esterification reaction.

The binding activity of the second portion to the polynucleic acid can be evaluated, for example, by measuring the number of living cells of HIV-infected cells in the case of RNA binding peptides (Example 1). Further, for example, the binding activity between the above RNA-binding peptide and RNA can be evaluated by relative quantification with a known peptide based on the expression level of the reporter gene in the following KAN system.

A preferred embodiment of the polynucleic acid binding substance of the present invention is, for example,

ClAc-GABA-RRDRRRLRQRARRRAAAA (SEQ ID NO: 2 9)

It is.

3 Second part.

In the present invention, the second portion has a heterogeneous or more selected from the group consisting of peptides, compounds, and nucleic acids. In the present invention, the second part preferably comprises a peptide.

(1) Peptides

In the present invention, the peptide (including the polypeptide) contained in the second part may be any peptide that can bind to the target polynucleic acid. The peptide contained in the second part of the present invention is preferably a peptide having an activity of binding to RNA, that is, an RNA binding peptide.

(i) RNA-binding peptide

In the present invention, the RNA-binding peptide includes, for example, 15 amino acid sequences rich in arginine and 23 amino acid sequences as a backbone, and has an activity of binding to the Rev response element (RRE) of HIV. It is a peptide that has HIV, and the growth of HIV can be suppressed by contacting the peptide with HIV. The peptide contained in the second part of the present invention has at least the following formula I: RQRX (I)

(X represents an amino acid residue other than R.)

Or a derivative thereof, or a salt thereof. In the above formula, R and Q are amino acid one-letter codes, and represent arginine (Arg) and glutamine (Gin), respectively (the same applies to other amino acids). X represents an amino acid residue other than arginine.

The peptide contained in the second part of the present invention comprises the amino acid sequence of the above formula, and preferably has a 12-23 amino acid sequence. Some amino acids in it form arginine-rich domains (called “arginine-rich domains”). For example, in a peptide having 23 amino acid residues, the 5th to 19th amino acids form an arginine-rich domain. The peptide contained in the second part of the present invention preferably contains 9 to 11 residues of arginine. The arginine rich domains of several RNA binding proteins have been shown to bind with high affinity and specificity to their similar RNAs. Since the sequence is simple and the structure is diverse, arginine-rich domain can be used as an effective tool for identifying novel RNA-binding peptides. The peptides contained in the first and second parts of the present invention can be obtained from a peptide library having an extremely large number of combinations by a screening technique called a kanamycin anti-termination system (referred to as “KAN system”).

The KAN system means an assembly that detects the interaction between RNA and peptide using the function of the kanamycin resistance gene in the cell (Hadas Peled-Zehavi, et al., RNA 9, 252-261, 2003).

Specifically, oligo DNA corresponding to the target RNA (here, “corresponding oligo DNA” means oligo DNA designed to have the same sequence as the target sequence), and kanamycin as a reporter gene An N reporter plasmid incorporating a resistance gene and an N expression plasmid incorporating a peptide-encoding oligonucleotide are introduced into the cell. RNA and peptides in the cell When interacted, the kanamycin resistance gene is transcribed and translated, and the cell acquires kanamycin resistance as a signal when the target RNA and peptide contact. For the cells, yeast, E. coli and the like can be used. When E. coli is used, peptides that interact with RNA appear in E. coli that survived in the medium containing kanamycin.

The amino acid sequences of the peptides contained in the second part of the present invention obtained by the KAN system are exemplified in the following SEQ ID NOs: 1 to 27, but the peptides contained in the second part of the present invention are exemplified. As long as has an RNA binding activity (activity that binds to the RRE of HIV), one or several amino acids of the amino acid sequence may have mutations such as deletion, substitution, addition, etc. Called "mutants"). For example, one or several amino acid sequences represented by SEQ ID NOs: 1-27, preferably 1-9, more preferably 1-5 amino acids may be deleted. SEQ ID NOs: 1-2 1 or several, preferably 1 to 9, more preferably 1 to 5 amino acids may be added to the amino acid sequence represented by 7 or the amino acid sequence represented by SEQ ID NOs: 1 to 27 One or several, preferably 1 to 9, more preferably 1 to 5 amino acids may be substituted with other amino acids.

17: MASARRRRRRRRRQRARRRAAAA (SEQ ID NO: 1)

39: MASRDRTRRRRTRQRSRRRAAAA (SEQ ID NO: 2)

42: MANDRRRRRRTRQRDRRRNAAAA (SEQ ID NO: 3)

11: MASLRRNRRRSRQRARRREAAAA (SEQ ID NO: 4)

26: MALLPRRERRRRRQRERRRAAAA (SEQ ID NO: 5)

8: MAVATRRERRRRRQRTRRRAAAA (SEQ ID NO: 6)

2-28: MASDPRRQRRRRRQRARRRAAAA (SEQ ID NO: 7)

2-07: MANPARRARRRCRQRERRRAAAA (SEQ ID NO: 8)

2-06: MAILARRQRRRGRQRERRRAAAA (SEQ ID NO: 9)

2- 04: MAQRERRERRRERQRNRRKAAAA (SEQ ID NO: 1 0)

3- 07: MGANARRRRRRARQRARRRGGG (SEQ ID NO: 1 1)

3-14: MGLSPRRDKRRR QRARRRGGG (SEQ ID NO: 1 2) 3-01: MGPHRRQRRRDRQRRRRRAAA (SEQ ID NO: 1 3)

3-12: MGLLRRDRRRQRQRQRRRGGG (SEQ ID NO: 1 4)

DRRRLRQRARRR (SEQ ID NO: 15)

DRREARQRARRR (SEQ ID NO: 1 6)

ERRRNRQRARRR (SEQ ID NO: 1 7)

D RRSRQRARRR (SEQ ID NO: 1 8)

HRRRLRQRARRR (SEQ ID NO: 1 9)

IRRRNRQRARRR (SEQ ID NO: 2 0)

LRRRARQRMRRR (SEQ ID NO: 2 1)

DRRRLRQRLRRR (SEQ ID NO: 2 2)

IRRRLRQRARRR (SEQ ID NO: 2 3)

ERRRIRQRARRR (SEQ ID NO: 2 4)

TRRRLRQRARRR (SEQ ID NO: 2 5)

HRRRLRQRLRRR (SEQ ID NO: 2 6)

DRRRRRQRIRRR (SEQ ID NO: 27) The binding activity between the peptide contained in the second part of the present invention and RNA is determined based on the expression level of the reporter gene in the KAN system and relative quantification with a known peptide. It can be measured by performing and evaluating. The dissociation constant at this time is 100ρΜ ~ ΜρΜ.

In the present invention, in addition to the above peptides, derivatives thereof can also be used. “Inducer” is derived from the peptide included in the second part of the present invention, and the number of amino acids is reduced to 3 or more amino acids, or some amino acids include unnatural products. Is substituted with the amino acid. The derivative may be a part of a natural product modified or a peptide containing a modified residue synthesized by chemical synthesis.

The peptides included in the second part of the present invention include those in which a part of the amino acid sequence is chemically modified. “Chemical modification” refers to the reaction of a chemical reagent with a protein, mainly changing the chemical structure of the amino acid residue side chain. For example, the second of the present invention For example, a method of reacting a reagent (for example, polyethylene glycol) that specifically modifies an amino acid that is expected to be present in the active site of the peptide or in the vicinity of the active site is employed. You can do an affinity label. Chemical modifications also include amino acids that are methylated at the amino acid carbon. Chemical modification methods are well known in the field (Sonori Ohno * Yu Kanaoka, Fumio Sakiyama, Hiroshi Maeda, Biochemical Experiment 12, Chemical Modification of Proteins (above), Academic Publishing Center).

The modified portion of the peptide containing a chemically modified amino acid sequence does not affect the peptide's original activity but acts as another effect (Yamaguchi, H. et al., Biosci. Biotechnol. Biochem. , 67 (10), 2269-2272, 2003).

Whether mutations such as substitutions and deletions have been introduced can be confirmed by sequencing amino acid sequences, molecular evolutionary engineering, and structural analysis using X-rays and NMR.

The derivative of the peptide included in the second part of the present invention also includes its retroenantiomer. The “retroenantiomer” means that the amino acid sequence of the peptide is reversed left and right (forms an enantiomer). In other words, it means that the N-terminus of the peptide is the C-terminus, the C-terminus is the N-terminus, and each amino acid is a sequence composed of D amino acids. Such retroenantiomers are also included in the present invention as long as they have RNA binding activity. For example, the retroenantiomer of the amino acid sequence shown in SEQ ID NO: 15 is RRRARQRLRRRD (SEQ ID NO: 28). The same applies to the amino acid sequences of other peptides.

Furthermore, the present invention relates to 65% or more, preferably 70% or more, more preferably 80% or more, more preferably 80% or more of the amino acid sequence constituting the peptide (SEQ ID NOs: 1 to 27), a variant thereof, or a derivative thereof. Peptides containing 90% or more, most preferably 95% or more, derivatives thereof or salts thereof can also be used. Examples of the 65% region include the 5th to 19th (Argyyunrich domain) regions of the sequences shown in SEQ ID NOs: 1 to 27, and the like. In addition, among the amino acid sequences shown in SEQ ID NOs: 1 to 27, the regions of the third to the 19th, the first to the 19th, the third to the 23rd as regions of 70% or more, 80% or more and 90% or more, respectively. Example area A region of 22 amino acids which are continuous as a region of 95% or more can be exemplified.

In addition to the amino acid sequences shown in SEQ ID NOs: 1 to 27, an amino acid sequence in which two arginines (R) are added to the N-terminal side of these amino acid sequences (an amino acid sequence having 14 amino acid residues) ) Is also contained in the peptide included in the second part of the present invention.

As described above, when the amino acid sequence of the peptide contained in the second part of the present invention is determined, DNA encoding the amino acid sequence is constructed and expressed, or the peptide is It can be obtained by chemical synthesis.

The peptide salt contained in the second part of the present invention is preferably a physiologically acceptable acid addition salt or basic salt. Examples of acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid, or acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citrate, apple And salts with organic acids such as acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid. Basic salts include, for example, salts with inorganic bases such as sodium hydroxide, hydroxy hydroxide, ammonium hydroxide and magnesium hydroxide, or organic compounds such as caffeine, piperidine, trimethylamine and pyridine. Examples include salts with bases.

Salts can be prepared using a suitable acid such as hydrochloric acid or a suitable base such as sodium hydroxide. For example, it can be prepared by treatment using standard protocols in water or in a liquid containing an inert water-miscible organic solvent such as methanol, ethanol or dioxane.

(ii) Peptide chemical synthesis

When the peptide included in the second part of the present invention is chemically synthesized, it can be synthesized by a well-known method of peptide synthesis. Examples include the azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, carboimidazole method, and redox method. In addition, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. Commercially available peptide synthesizer (Shimadzu Corporation You can also use PSSM-8, etc.).

After the reaction, the peptide contained in the second part of the present invention can be purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.

(iii) a polynucleotide encoding a peptide

The polynucleotide encoding the peptide contained in the second part of the present invention can be obtained by designing the peptide used in the present invention by genetic engineering. For example, a base sequence may be designed and synthesized based on the amino acid sequence of the peptide contained in the second part of the present invention. Examples of the polynucleotide include DNA and RNA, and DNA is preferred.

To obtain a mutant peptide by genetic engineering, a polypeptide encoding the amino acid sequence of SEQ ID NOs: 1 to 27 is prepared by a site-directed mutagenesis method well known in the art. can do. A commercially available kit for site-directed mutagenesis (for example, TaKaRa Site-Directed Mutagenesis System (Mutan'K, Mutan'Super Express Km, etc .: manufactured by Tacarava)).

(iv) Recombinant vectors, transformants and peptides

A recombinant vector for protein expression can be obtained by ligating the above polynucleotide to an appropriate vector, and a transformant introduces the above recombinant vector into the host so that the target gene can be expressed. (Sambrook J and Russel D. Molecular Cloning, A Laboratory Manual, 3rd edition, CSHL Press, 2001) _d

As the vector, a phage or plasmid capable of autonomously growing in a host microorganism is used. Examples of the plasmid DNA include plasmids derived from Escherichia coli, Bacillus subtilis, or yeast. Examples of the phage DNA include L phage. In addition, animal virus and insect virus vectors can also be used.

For the production of a recombinant vector, the purified DNA may be cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site of an appropriate vector DNA, etc., and ligated to the vector. The host used for transformation is not particularly limited as long as it can express the target gene. For example, bacteria (E. coli, Bacillus subtilis, etc.), yeast, movement Examples include physical cells (COS cells, CHO cells, etc.) and insect cells.

Methods for introducing a recombinant vector into a host are known, and include any method (for example, a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, a calcium phosphate method, a ribofusion method, etc.). . The peptide contained in the second part of the present invention can also be obtained by culturing the transformant and collecting it from the culture. “Culture” means any of (a) culture supernatant, (b) cultured cells or cultured cells, or crushed materials thereof.

Culture methods are well known in the art (see Sambrook et al., Molecular Cloning, supra).

When the target peptide is produced in cells or cells after culturing, proteins are extracted by disrupting the cells or cells. If the target protein is produced outside the cells or cells, use the culture solution as it is, or remove the cells or cells by centrifugation or the like. Then, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, It is possible to isolate and purify the peptide.

In the present invention, peptide synthesis by in vitro translation can be employed. In this case, two methods can be used: the method of making RNA RNA and the method of making DNA DNA (transcription Z translation). Examples of the cage RNA include the polynucleotide described in (iii) above. The cage DNA includes the polynucleotide having a promoter and a ribosome binding site upstream of the translation initiation site, or the translation initiation site. And a polynucleotide in which a promoter necessary for transcription is incorporated upstream. As an in vitro translation system, a commercially available system such as ExpresswayTM system (Invitrogen), PURESYSTEM (registered trademark; Postgen Institute), TNT system (registered trademark; Promega), etc. can be used. After peptide synthesis by an in vitro translation system, the desired peptide is isolated and purified by combining the above general biochemical methods alone or in combination. Can.

(2) Compound

The compound contained in the second part of the present invention may be any compound that can bind to the target polynucleic acid. Examples thereof include one or more compounds contained in natural or synthetic low molecular weight compound libraries and plant or animal extracts. These compounds may be novel compounds or known compounds.

A compound that binds to the target polynucleic acid can be obtained, for example, by a binding assay between the target polynucleic acid and the compound. In this case, for example, the compound bound to the polynucleic acid can be selected by bringing a compound library or the like into contact with a carrier on which the target polynucleic acid is immobilized. In addition, a compound that binds to a target polynucleic acid can be selected using a part of the KAN system. In this case, the compound that has been brought into contact with the cell that has acquired kanamycin resistance when the compound is brought into contact with the cell into which the aforementioned N reporter plasmid has been introduced can be selected as a compound that binds to the target polynucleic acid. .

As long as the compound contained in the second part of the present invention has an action of binding to the target polynucleic acid, it may form a salt or a hydrate thereof. Alternatively, a compound identified by CADD (Computer Aided Drug Design) is also included in the compound included in the second part of the present invention as long as it has an action of binding to the target polynucleic acid.

Preferable examples of the compound contained in the second part of the present invention include, for example, an aminoglycoside, a heterocyclic amine, a salt thereof, or a hydrate thereof.

(3) Nucleic acid

The nucleic acid contained in the second part of the present invention may be any nucleic acid as long as it has an action of binding to the target polynucleic acid, and is not particularly limited, but is preferably an antisense oligonucleotide. Or siRNA. The present invention The nucleic acid contained in the second part of the nucleic acid includes, for example, a nucleic acid having a base sequence complementary to the binding partner (target) polynucleic acid, a nucleic acid having a base sequence that binds to the binding partner polynucleic acid under stringent conditions, etc. Can be mentioned. Here, the “stringent conditions” are, for example, conditions at the time of washing after hybridization, for example, a salt concentration of 300 to 2000 mM, a temperature of 40 to 75 ° C., preferably a salt concentration. It means the condition of 600-900mM and temperature of 65 ° C. The nucleic acid contained in the second part of the present invention is a polynucleotide that specifically hybridizes under such stringent conditions as the base sequence of the binding partner polynucleic acid and 80% The nucleic acid having homology of 90% or more, more preferably 95% or more is preferable.

The nucleic acid contained in the second part of the present invention may be a nucleic acid having a novel sequence or a nucleic acid having a known sequence.

An antisense oligonucleotide is a nucleic acid having a sequence complementary to a target mRNA and is used in the antisense method. Antisense oligonucleotides can be appropriately designed and synthesized by those skilled in the art according to the target mRNA.

siRNA is a short-chain RNA produced by processing by Dicer in a cell, and is used as an effector of RNAi.

The design criteria for siRNA are as follows, and those skilled in the art can appropriately design and synthesize siRNA.

(a) Select the region downstream of the start codon of the gene containing the binding partner polynucleic acid. The sequence is not particularly limited as long as it is a sequence downstream from the start codon, and any region can be used as a candidate.

(b) From the selected region, a sequence of 15 to 30 bases, preferably 18 to 25 bases starting with aa is selected, and a sequence whose GC content is, for example, 20 to 80% is selected.

The nucleic acid contained in the second part of the present invention can be synthesized by an automatic nucleic acid synthesizer. Alternatively, a person skilled in the art introduces the nucleic acid contained in the second part of the present invention into a vector such as a plasmid, and uses the host vector system such as E. coli for the present invention. Can produce nucleic acids contained in the second part of

3. Pharmaceutical composition containing polynucleic acid binding substance

The polynucleic acid binding substance of the present invention can be used as a pharmaceutical composition. When the second part contained in the polynucleic acid-binding substance of the present invention has an action of suppressing or enhancing the gene expression of the target polynucleic acid, the pharmaceutical composition of the present invention results from an increase or decrease in the expression of the gene. It can be used as a therapeutic or prophylactic agent for diseases. In the present invention, the disease may be a single disease, a concurrent disease, or a disease other than the above, all of which are intended for use with the peptide of the present invention. be able to.

In addition, when the second part contained in the polynucleic acid-binding substance of the present invention has an activity of binding to, for example, HIV RRE, the pharmaceutical composition of the present invention is used as an HIV growth inhibitor or an infection preventive agent. be able to.

When the pharmaceutical composition of the present invention is used as an HIV growth inhibitor or infection preventive agent, it has a specific purpose of treating or preventing AIDS (acquired immune deficiency syndrome) patients and HIV virus positive healthy individuals. Can be used. It can also be used for the prevention of infection of healthy subjects.

The pharmaceutical composition of the present invention can be systemically or locally administered orally or parenterally. When the pharmaceutical composition of the present invention is orally administered, it may be any of tablets, force-pellants, granules, powders, pills, troches, liquids for internal use, suspensions, emulsions, syrups, etc. Alternatively, it may be a dry product that is redissolved when used. In addition, when the pharmaceutical composition of the present invention is administered parenterally, a pharmaceutical form such as intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. can be selected. In the case of injectable preparations, they are provided in the form of unit dose ampoules or multi-dose containers.

These various preparations include excipients, bulking agents, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, preservatives that are commonly used in pharmaceutical preparations. A flavoring agent, a soothing agent, a stabilizer, a tonicity agent and the like are appropriately selected and can be produced by a conventional method. The above-mentioned various preparations may contain a pharmaceutically acceptable carrier or additive together. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, sodium anoleate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, strong zein Gelatin, agar, glycerin, propylene dallicol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, ratatose and the like. The additive to be used is appropriately selected or combined from the above according to the dosage form of the present invention.

The dosage of the pharmaceutical composition of the present invention varies depending on the age of administration subject, administration route, and number of administrations, and can be varied over a wide range. The effective amount administered as a combination of an effective amount of the polynucleic acid-binding substance of the present invention and an appropriate diluent and a pharmacologically usable carrier is 10 to 1000 mg / body per kg body weight at a time, preferably The dose in the range of 50 to 500 mg / body can be selected, and it is administered once or more in one to several times a day. Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Anti-HIV activity test

(1) Method

Pauwels et al "J. Virol. Methods, 20; 309-321,1988, Nakashima et al" Antimicrob. Agents Chemother., 36; 1249-1255, 1992, Nakashima et al., Antiviral Chemist. Chemother ., 6; 271-280, 1995.

Specifically, HIV-infected MT-4 cells (2.5 × 10 ⁴ cells / well, ΜΟΙΌ.ΟΙ) were added to a 96-well microtiter plate with various concentrations of test substances immediately after infection.

In order to know the cytotoxicity of the test substance to ΜΤ-4 cells, the virus-uninfected cells were also cultured with the test substance at the same concentration as the test area. Cultivation was performed with serum. After culturing at 37 ° C for 5 days in a CO ₂ incubator, the number of viable cells was measured by the MTT method. Antiviral activity is the concentration that protects 50% of cytotoxicity due to HIV infection (EC ₅ o; 50% effective concentration), and cytotoxicity is the 50% cytotoxic concentration (CC ₅₀ ; 50% cytotoxic concentration) of the test substance. Expressed. Effectiveness coefficient (Selectivity Index; SI) was calculated as CC ₅₀ / EC ₅₀ .

The test substance used was

Contole peptide 1: _suc -RRDRRRLRQRARRRAAAA-OH (SEQ ID NO: 30)

Test peptide 5: ClAc-GABA-RRDRRRLRQRARRRAAAA-OH (SEQ ID NO:

3 1)

It is. “Suc” refers to the protecting group succinyl.

(2) Results

The results are shown in Table 1.

Table 1 Anti-HIV activity test results

From the results in Table 1, it was shown that test peptide 5 to which an electrophilic group and a spacer were added improved the pharmacological activity by binding the electrophilic group and the spacer. Industrial applicability

The present invention provides a polynucleic acid binding substance comprising an electrophilic group and one or more selected from the group consisting of peptides, compounds and nucleic acids. Since the polynucleic acid binding substance of the present invention contains an electrophilic group, it has a high binding activity to polynucleic acid. Therefore, it can be said that the polynucleic acid-binding substance of the present invention is useful as a pharmaceutical composition. In particular, when the peptide contained in the polynucleic acid binding substance of the present invention is an RNA binding peptide, it is used for treatment or prevention of RNA viral diseases. Can sequence table free text

SEQ ID NOs: 1-39: synthetic peptides

SEQ ID NO: 29: An electrophilic group spacer 1 is bonded to the first amino acid. SEQ ID NO: 30: Succinyl is bonded to the first amino acid. .

SEQ ID NO: 31: An electrophilic group spacer 1 is bonded to the first amino acid.

Claims

The scope of the claims

1. A substance that binds to a polynucleic acid, comprising a first part having an electrophilic group, and a second part having one or more selected from the group consisting of peptides, compounds and nucleic acids. A characteristic polynucleic acid binding substance.

2. The polynucleic acid-binding substance according to claim 1, wherein the polynucleic acid is RNA.

3. The polynucleic acid-binding substance according to claim 2, wherein the RNA contains an RRE sequence site.

4. The polynucleic acid binding substance according to claim 1, wherein the second part comprises a peptide.

5. The peptide has the following formula I:

R-Q R-X (I)

(X represents an amino acid residue other than R.)

5. The polynucleic acid-binding substance according to claim 4, which is a peptide comprising the amino acid sequence represented by

6. The polynucleic acid-binding substance according to claim 4, wherein the peptide is the following peptide (a) or (b), a derivative thereof, or a salt thereof.

(a) Formula I:

R-Q-R-X (I)

(X represents an amino acid residue other than R.)

(b) Formula I:

R-Q-R-X (I)

(X represents an amino acid residue other than R.)

An amino acid sequence having a length of 12 to 23 residues including the amino acid sequence represented by the above, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and have RNA binding activity Peptide

7. The polynucleic acid-binding substance according to claim 5 or 6, wherein R contains at least 9 to: L1 residue.

8. The polynucleic acid-binding substance according to claim 4, wherein the peptide is the following peptide (a) or (b), a derivative thereof, or a salt thereof.

(b) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NOs: 1 to 27, and

Peptides with RNA binding activity

9. The polynucleic acid-binding substance according to claim 1, wherein the electrophilic group is a group represented by the following formula.

(In the formula, Z represents a halogen atom.)

10. The polynucleic acid binding substance according to claim 1, wherein a spacer is provided between the first part and the second part.

1 1. A pharmaceutical yarn composition comprising the polynucleic acid binding substance according to any one of claims 1 to 10.

12. The pharmaceutical composition according to claim 11, which is an HIV growth inhibitor.

1 3. The pharmaceutical composition according to claim 11, which is a preventive agent for HIV infection.

14. The pharmaceutical composition according to claim 11, which is a preventive or therapeutic agent for AIDS.