JP2006036711A - METHOD OF INHIBITING DNase ACTIVITY OF DNase gamma, METHOD OF SCREENING INHIBITOR INHIBITING DNase ACTIVITY OF DNase gamma, AND DNase gamma INHIBITOR - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of inhibiting DNase activity of DNase γ, a method of screening inhibitors inhibiting the DNase activity of DNase γ, and an inhibitor inhibiting the DNase activity of the DNase γ. <P>SOLUTION: The DNase activity of the DNase γ can be inhibited by bonding a substance to a domain corresponding to a DNA bonding pocket where one chain DNA bonding to the enzyme activity center of DNase I and one chain DNA on the different side, when the DNase I cuts DNA while dissociating two chain DNA. By utilizing the inhibiting method like this, such a screening that selecting the compound bonding to the DNase γ from a compound library and confirming whether it is possible or not to inhibit the DNase activity of the DNase γ. A substance obtained by this can inhibit the DNase activity of the DNase γ. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an inhibition method for inhibiting DNase γ DNase activity, a screening method for an inhibitor that inhibits DNase γ DNase activity, and a DNase γ inhibitor obtained by the screening method.

  Apoptosis is a concept corresponding to necrosis, which is phenomenologically characterized by cell death characterized by cell shrinkage, chromatin condensation, DNA fragmentation, and the like. Apoptosis plays an important role not only in the morphogenesis and development of the nervous system network during development, but also in the removal of abnormal cells in the adult, maintenance of homeostasis by the endocrine system, and establishment of the immune system. Abnormal apoptosis causes diseases such as cancer, autoimmune disease, neurodegenerative disease hepatitis, AIDS and the like. In this way, apoptosis is one of the most important basic functions of cells, so it is important to be always taken into consideration in a wide range of medical fields ranging from the elucidation of various diseases to the development of appropriate treatments. It is a phenomenon.

  DNase I, DNase X, DNaseγ, CAD / DFF40, Endonuclease G, DNase II and the like have been reported as enzymes involved in DNA fragmentation, which is one of the methods for detecting apoptosis. However, as to whether these DNases actually work in the living body and how they are used properly, that is, regarding the types of cells, differentiation states, apoptosis-inducing stimuli, etc., where each DNase is specifically involved, Has not been clarified yet.

The three-dimensional structure of DNase I was revealed by X-ray crystallography performed using a protein co-crystallized with DNA (see Non-Patent Document 1). In this three-dimensional structure, of the dissociated DNA double strands, the active center to which one DNA strand binds and the domain to which the other DNA strand binds (hereinafter this domain is called DNA binding pocket) are identified. (See Non-Patent Document 1). With regard to the active center, it has been clarified that the activity of DNase γ disappears by introducing a mutation into the amino acid sequence that constitutes the active center (see Non-Patent Document 2). The relationship with was not always clear.
J. Mol. Biol. 222, 645-67, 1991 Understanding Apoptosis Understanding Experimental Medicine Series-Basics & Topics (Editor: Junichi Tanuma, Publisher: Yodosha, ISBN: 4897069882) p. 69

  To elucidate the role of DNaseγ in apoptosis and the specificity of DNaseγ-dependent apoptosis, specifically inhibiting DNaseγ's DNase activity is one strategy. However, at present, there is no effective activity inhibiting substance or inhibition method for that purpose, and development thereof is required.

  Accordingly, an object of the present invention is to provide a method for inhibiting the DNase activity of DNaseγ, a screening method for an inhibitory substance that inhibits the DNase activity of DNaseγ, and a substance that inhibits the DNase activity of DNaseγ.

  In order to solve the above problems, the present inventors first predicted the three-dimensional structure of DNaseγ based on the DNase I co-crystal structure. In the three-dimensional structure of DNase I, when DNase I dissociates double-stranded DNA and cleaves the DNA, the other DNA strand binds to the enzyme active center of DNase I that binds to the DNA strand that is to be cleaved. Although a DNA binding pocket exists, the present inventors have identified a domain corresponding to the DNA binding pocket of DNase I in the three-dimensional structure of DNaseγ. In the present specification, the domain of DNaseγ corresponding to the DNA binding pocket of DNase I is referred to as the DNA binding pocket of DNaseγ regardless of whether it actually binds to DNA.

  Subsequently, a substance having a high binding affinity with the DNA binding pocket of DNaseγ is selected from the compound library, and then, by experimentally examining whether the selected compound can inhibit the DNase activity of DNaseγ, A substance that inhibits the DNase activity of DNaseγ (hereinafter referred to as a DNaseγ inhibitor) has been found and the present invention has been completed.

  That is, the inhibition method for inhibiting the DNase activity of DNaseγ according to the present invention includes binding a substance to the DNA binding pocket of DNaseγ in order to cause a steric binding disorder. This substance includes one or more amino acid residues in the S1 domain (see SEQ ID NO: 2) consisting of Glu13, Ser14, Asp42, Ser43, and Asn44 of human DNaseγ (see SEQ ID NO: 1), and Ser10 and Phe11 of DNaseγ. , Gly12, Glu39, Ile40, and Lys41 may bind to one or more amino acid residues of the S2 domain (see SEQ ID NO: 3), but DNaseγ Arg72, Thr77, Tyr78, Lys79 , Glu80, and Gln81 may bind to one or more amino acid residues of the S3 domain (see SEQ ID NO: 4).

As a specific substance, for example, a substance containing any compound selected from the group consisting of the following formulas (1) to (4) or a pharmacologically acceptable salt thereof as an active ingredient can be used.

  The method for screening an inhibitor that inhibits the DNase activity of DNaseγ according to the present invention includes a primary screening for selecting a substance that binds to the DNA binding pocket of DNaseγ from a compound library, and the selected substance is the DNase activity of DNaseγ. Secondary screening to confirm whether it can be inhibited.

Furthermore, the DNaseγ inhibitor according to the present invention contains any compound selected from the group consisting of the following formulas (1) to (4) or a pharmaceutically acceptable salt thereof as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the inhibition method which inhibits the DNase activity of DNaseγ, the screening method of the inhibitor which inhibits the DNase activity of DNaseγ, and the substance which inhibits the DNase activity of DNaseγ can be provided.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Inhibition method of inhibiting DNase activity of DNaseγ ==
Although the DNA binding pocket of DNaseγ has not yet been elucidated by X-ray analysis or NMR (Nuclear Magnetic Resonance) method, it is expected to be a DNA binding site at the time of DNA cleavage like DNaseI. Therefore, it is considered that a substance that binds to the DNA binding pocket has an inhibitory effect on the DNase activity of DNaseγ by causing steric binding damage to the stable binding of DNA to DNaseγ.

  Therefore, DNase activity of DNaseγ can be inhibited by binding a substance to the DNA binding pocket of DNaseγ in order to cause a steric binding disorder. Any substance can be used as long as it can inhibit the DNase activity of DNaseγ, a compound that binds to the DNA binding pocket by hydrogen bonding, an antibody that has an epitope around the DNA binding pocket, and binds firmly to the DNA binding pocket. Examples include modified single-stranded DNA.

== Screening method for inhibitors inhibiting DNase activity of DNaseγ ==
By using this inhibition method for inhibiting the DNase activity of DNaseγ, an inhibitory substance that inhibits the DNase activity of DNaseγ can be screened.

  First, as a primary screening, a substance that binds to a DNA binding pocket of DNaseγ is selected from a compound library. It is preferable to select a substance having a high binding affinity (for example, hydrogen bond, hydrophobic interaction, etc.) to the DNaseγ of the substance. As a secondary screening, an inhibitor that actually inhibits DNase activity can be identified by experimentally confirming whether the substance selected in the primary screening can inhibit the DNase activity of DNaseγ.

  The primary screening can be performed using a Docking program on a computer, for example. In addition, as a compound library, databases, such as ChemACX, Maybridge Catalog and the Asinex database, Rare Chemical Library, World Drug Index, Cambridge Crystallographic Database, can be used, for example.

  Alternatively, a substance that binds to the DNA binding pocket of DNaseγ can be selected experimentally. For example, an affinity column carrying a DNA binding pocket portion of DNaseγ expressed in E. coli or the like may be used, and a compound that binds to the column may be selected from the compounds belonging to the compound library.

  In the secondary screening, it is experimentally confirmed whether the substance actually has an inhibitory effect on DNase activity of DNaseγ. Any confirmation method can be used as long as it can confirm the inhibitory effect of DNaseγ on the DNase activity, whether in vitro or in vivo.

  For example, it is possible to confirm whether or not this substance can inhibit DNase activity in vitro by adding the substance selected in the primary screening to a solution containing DNaseγ and an appropriate length of DNA and causing an enzyme reaction.

  Alternatively, DNaseγ-dependent apoptosis may be induced in cultured cells, and the substance selected in the primary screening may be added to the medium to confirm whether this substance can inhibit DNA fragmentation. In this case, it is desirable to confirm that this substance does not have a detrimental effect on other processes of apoptosis.

== Inhibitor that inhibits DNaseγ DNase activity ==
As described in the Examples, the following compound (1) was obtained as an inhibitor that inhibits the DNase activity of DNaseγ by the above screening method. Further, when the binding mode between the compound (1) and the DNA binding pocket of DNaseγ was examined, as shown in FIG. 1, the compound (1) was composed of S1 of DNaseγ (Glu13, Ser14, Asp42, Ser43, and Asn44). ), S2 (consisting of Ser10, Phe11, Gly12, Glu39, Ile40, and Lys41), S3 (consisting of Arg72, Thr77, Tyr78, Lys79, Glu80, and Gln81) and hydrogen bonds It became clear to do.

Similarly, the following compound (2) was obtained from the compound library by the above screening method. Examination of the binding mode between the compound (2) and the DNA binding pocket of DNaseγ revealed that compound (2) binds to amino acid residues in domains such as S1 and S2 of DNaseγ, as shown in FIG. Became.

  Like these compounds, S1 (consisting of Glu13, Ser14, Asp42, Ser43, and Asn44), S2 (consisting of Ser10, Phe11, Gly12, Glu39, Ile40, and Lys41), S3 in the DNA binding pocket of DNaseγ. Compounds that hydrogen bond with amino acid residues in domains such as Arg72, Thr77, Tyr78, Lys79, Glu80, and Gln81 can stably bind to the DNA binding pocket and cause steric binding damage. Can do.

Further, similarly, the following compounds (3) and (4) that inhibit the DNase activity of DNaseγ were obtained from the compound library by the above screening method.

  Examples of the present invention will be described in detail below.

[Example 1] Prediction of steric structure of DNaseγ (1) Based on the DNase I crystal structure (PDB code: 2DNJ), an initial structure of DNaseγ was constructed by homology modeling (see FIG. 3). Note that Modeller (http://salilab.org/modeller/modeller.html) was used as software for the homology modeling method.
(2) Since the three-dimensional structure of DNase I used as a template is a co-crystal with DNA, a DNaseγ / DNA complex structure was constructed based on the complex structure.
(3) The structure of the DNaseγ / DNA complex structure was optimized using Amber, a molecular dynamics software, to obtain a final three-dimensional structure.
(4) The validity of the three-dimensional structure of the predicted DNaseγ / DNA complex was evaluated using Verify3D.

  As a result of the evaluation using Verify3D, the score is 121.41, and the validity standard value of the three-dimensional structure (S = exp (−0.83 + 1.008ln (L)); L is the number of residues (L = 256)) is 116.7 From the above, it was judged that the predicted three-dimensional structure of the DNaseγ / DNA complex was appropriate. Moreover, the domain (S1, S2, S3) of DNaseγ corresponding to the DNA binding pocket of DNase I was predicted from the three-dimensional structure of the predicted DNaseγ / DNA complex (see FIG. 2). The amino acids contained in these domains are S1 (Glu13, Ser14, Asp42, Ser43, Asn44), S2 (Ser10, Phe11, Gly12, Glu39, Ile40, Lys41), S3 (Arg72, Thr77, Tyr78, Lys79, Glu80, Gln81 )Met.

[Example 2] Primary screening of DNaseγ inhibitor by Docking Program Next, using the structure of DNaseγ predicted in Example 1, using the Docking program (Autodock 3.0), the compounds contained in the compound library and DNA of DNaseγ By docking the binding pocket and calculating the binding affinity score, a substance that can be an inhibitor of DNaseγ (compounds (1) to (4) below) was selected. In addition, ChemACX was used as the compound library, and all parameters were set as defaults.

[Example 3] Inhibitory effect of DNaseγ on DNase activity (secondary screening)
In order to confirm whether the compounds (1) to (4) selected according to Example 2 can inhibit the DNase activity of DNaseγ, the compounds (1) and (2) were obtained from Sigma Corporation as a compound (3). Was purchased from Fluka and Compound (4) was purchased from Molecular Probes, and the inhibitory effect on DNA cleavage activity by DNaseγ was examined in vitro.

(1) A compound of each concentration (any of compounds (1) to (4)) and DNaseγ (final 8 × 10 ⁻⁴ Kunitz units / μl) are mixed with a reaction solution (50 mM Mops-NaOH (pH 7.2), 3 It was suspended in 45 μl of mM MgCl ₂ , 3 mM CaCl ₂ , 0.1 mg / ml BSA (bovine serum albumin)) and incubated at 37 ° C. for 30 minutes.
(2) Salmon sperm DNA (0.5 mg / ml) 5 μl was added and reacted at 37 ° C. for 20 minutes (total 50 μl).
(3) The reaction was stopped by adding 50 μl of 10% PCA.
(4) Left on ice for 20 minutes.
(5) Centrifugation was performed at 3000 rpm and 4 ° C. for 15 minutes.
(6) The supernatant was diluted with distilled water, and the acid-soluble DNA product decomposed by DNaseγ was measured for absorbance A ₂₆₀ , the activity of DNaseγ was calculated, and IC ₅₀ was determined.

The results are shown in Table 1.

  As shown in Table 1, it was revealed that all of the compounds (1) to (4) inhibit DNase activity of DNaseγ. In particular, Compound (1) and Compound (2) showed an excellent inhibitory effect.

[Example 4] Confirmation of DNA fragmentation inhibitory effect on apoptosis (secondary screening)
Next, it was examined whether the compound selected by Example 2 can suppress genomic DNA fragmentation in apoptosis of cultured cells.

(1) HeLa S3 cells in which endogenous DNaseγ expression is hardly detected and DNA fragmentation does not occur in response to various apoptotic stimuli in medium (Dulbecco's modified Eagle medium containing 10% FCS) in 3.5 cm 1 × 10 ⁵ seeds were sown in a dish.
(2) hDNaseγ obtained by subcloning Human DNaseγ cDNA into Not I Site of vector pcDNA3.1 / Myc-His (Invitrogen) to give cells DNaseγ-dependent apoptotic activity after overnight culture and medium change Were introduced into HeLa S3 cells (samples 3 to 6). The gene transfer was performed using FuGene6 (Roche).
(3) After further overnight culture and changing the medium, 0.5 μM staurosporine (STS; apoptosis inducer) (samples 2 to 6) and compound (1) (samples 3 to 6) at each concentration were added. And incubated for 24 hours.
(4) Cells are collected, treated with proteinase K (proteolytic enzyme) and RNase A (RNA degrading enzyme) to obtain an extract from the cells, and then the extract is electrophoresed on a 1.8% agarose gel. did. The results are shown in FIG.

The following is a summary of the processing for each sample.
(Sample 1) DNA only (Sample 2) Negative control (STS (+) DNaseγ (-))
(Sample 3) Positive control (STS (+) DNaseγ (+))
(Sample 4) (STS (+) DNaseγ (+) Compound (1) (10 μM))
(Sample 5) (STS (+) DNaseγ (+) Compound (1) (30 μM))
(Sample 6) (STS (+) DNaseγ (+) Compound (1) (100 μM))
As shown in FIG. 4, it was revealed that Compound (1) inhibited genomic DNA fragmentation by DNaseγ in a concentration-dependent manner, and almost completely at 100 μM.

  Furthermore, in order to confirm that compound (1) does not affect other processes of apoptosis, that is, processes other than genomic DNA fragmentation, PARP (poly (ADP-ribose) polymerase-1), which is an index of apoptosis, is used. The effect of compound (1) on cleavage was investigated. As described above, HeLa S3 cells into which hDNaseγ expression vector was introduced were treated with 0.5 μM staurosporine (STS; apoptosis inducer) and 100 μM compound (1) (sample 4), and then obtained by homogenizing the cells. Western blotting was performed using the extracted extracts. As a control, the same experiment was carried out on untreated cells (sample 1), cells treated only with compound (1) (sample 2), and cells treated only with STS (sample 3). The primary antibody is an anti-PARP monoclonal antibody (used in Wako No. 016-16831, 1: 500), and the secondary antibody is an anti-mouse IgG antibody (used by Promega, 1: 1000), Western-Blue ( Promega) was used to detect the signal.

  As shown in FIG. 5 (Sample 4), since PARP cleavage was detected even in the presence of Compound (1), Compound (1) had little effect on the initiation and progression of apoptosis, and was caused by DNaseγ. It was found to specifically inhibit genomic DNA fragmentation.

FIG. 3 is a view showing a binding mode between compound (1) and DNaseγ. FIG. 3 is a view showing a binding mode between compound (2) and DNaseγ. It is a figure which shows the three-dimensional structure of DNaseγ. FIG. 2 is a graph showing an effect of compound (1) inhibiting DNA fragmentation by DNaseγ. It is a figure which shows the result of having investigated the influence of the compound (1) with respect to the cutting | disconnection of PARP (poly (ADP-ribose) polymerase-1) which is a parameter | index of apoptosis.

Claims (7)

An inhibition method for inhibiting DNase activity of DNaseγ,
An inhibitory method comprising binding a substance to a DNA binding pocket of DNaseγ to cause a steric binding disorder. The inhibition method according to claim 1, wherein the substance contains a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient.
2. The substance according to claim 1, wherein the substance contains any compound selected from the group consisting of the following formulas (2) to (4) or a pharmacologically acceptable salt thereof as an active ingredient. Inhibition method.
  The substance comprises one or more amino acid residues of S1 domain consisting of Glu13, Ser14, Asp42, Ser43 and Asn44 of DNaseγ, and S2 domain consisting of Ser10, Phe11, Gly12, Glu39, Ile40 and Lys41 of DNaseγ. The method according to claim 1, wherein the method binds to one or more amino acid residues.   The inhibition method according to claim 4, wherein the substance further binds to one or more amino acid residues of the S3 domain consisting of Arg72, Thr77, Tyr78, Lys79, Glu80, and Gln81 of DNaseγ. A screening method for an inhibitor that inhibits DNase activity of DNaseγ,
A primary screening for selecting a substance that binds to the DNA binding pocket of DNaseγ from a compound library;
A secondary screening to confirm whether the selected substance can inhibit the DNase activity of DNaseγ;
A screening method comprising the steps of: A DNaseγ inhibitor comprising as an active ingredient any compound selected from the group consisting of the following formulas (1) to (4) or a pharmacologically acceptable salt thereof: