JP2009242247A - Human immunodeficiency virus infection inhibitor and aids-treating or preventing drug - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel human immunodeficiency virus infection inhibitor, and an aids-preventing and treating drugs. <P>SOLUTION: The human immunodeficiency virus infection inhibitor contains a compound represented by formula (I) or a pharmaceutically acceptable salt or hydrate thereof as an active ingredient. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a human immunodeficiency virus infection inhibitor, and a new type of AIDS preventive and therapeutic agent containing the inhibitor.

  Acquired immunodeficiency syndrome (AIDS; AIDS) is known to cause CD4 positive T cells to be destroyed by infection with human immunodeficiency virus (HIV), thereby significantly reducing the immune function of the host and causing various complications. ing. To date, AIDS drugs approved in Japan include nucleic acid reverse transcriptase inhibitors such as zidovudine (AZT), didanosine (ddI), and zalcitabine (ddC), and non-nucleic acid reverse transcription such as nevirapine, efavirenz, and delavirdine. Enzyme inhibitors and protease inhibitors such as indinavir (crixiban) and saquinavir. Although these drugs are effective for AIDS treatment, HIV-1 is apt to be mutated, and one problem is that drug-resistant strains appear in the course of treatment. A therapy using a combination of various drugs has an effect of reducing the appearance of drug-resistant strains. However, currently available therapeutic agents are limited to those targeting HIV reverse transcriptase and protease as described above, so there are limitations to multi-drug therapy. Therefore, the development of AIDS therapeutics for new molecular targets is expected.

One of the HIV-1 accessory genes, vpr, consists of 96 amino acids and encodes a 15 kilodalton (kDa) nucleoprotein that binds to viral particles. Primate lentiviruses such as HIV-1, HIV-2, and SIV all contain very well conserved Vpr-like proteins. Many biological functions of the Vpr protein have been reported in relation to HIV replication, for example, activating transcription of various retroviral LTRs, including HIV, or Vpr is a cell cycle G ₂ / M It stops at a period (for example, refer nonpatent literature 1 and 2). In cells arrested in the G ₂ / M phase by this Vpr function, viral replication increases about 2-fold, which is considered to play an important role in the growth of HIV. Furthermore, according to in vivo experiments using primates infected with SPR deficient in vpr, it has been reported that Vpr function is important for the development of acquired immunodeficiency (AIDS) ( For example, refer nonpatent literature 3).

  Thus, it has been proposed that Vpr is one target protein of anti-AIDS drugs. A cell line has been established in which cell cycle arrest induced by Vpr is regulated by a tetracycline promoter, and screening with this cell has shown that certain flavonoids (quercetin) inhibit Vpr protein function (eg, Non-patent document 2). It has also been reported that expression of Vpr protein in budding yeast suppresses yeast growth (see, for example, Non-Patent Document 4).

On the other hand, it has been reported that a compound isolated from a certain filamentous fungus inhibits the cell growth arresting ability of Vpr and that the compound is used as an AIDS therapeutic drug (see, for example, Patent Document 1).
Further, in the production of HIV-1 mRNA in a host cell, a substance that increases the production of mRNA group of 4 kb form and / or inhibits the activity of reducing the production of mRNA group of 2 kb form is a proliferation of HIV-1. It has been reported that the composition can be used as a pharmaceutical composition for suppressing the depression (see, for example, Patent Document 2).

Furthermore, the interaction between Vpr protein and importin α is important for HIV-1 infection of human immune cells (see, for example, Non-Patent Documents 5 and 6), and a compound that inhibits the binding between Vpr protein and importin α is used. It has been reported that a drug that inhibits HIV-1 infection of human immune cells can be obtained by screening (see, for example, Patent Document 3).
However, low molecular weight compounds that inhibit HIV-1 infection are not yet well known, and the development of drugs with excellent medicinal effects has been demanded.
Japanese Unexamined Patent Publication No. 2006-321759 International Publication No. 2005/032561 Pamphlet JP 2006-067994 JP Curr. Biol. Vol. 6, pp.1096-1103. (1996) Biochemical and Biophysical Research Communication Vol. 261, No. 2, pp.308-316. (1999) J. Virol. Vol. 69, pp.4807-4813. (1995) Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 2770-2774. (1995) Journal of Virology, May 2007, p. 5284-5293, Vol. 81, No. 10 Journal of Virology, March 2005, p. 3557-3564, Vol. 79, No. 6

  An object of the present invention is to find a novel AIDS therapeutic or prophylactic agent capable of inhibiting HIV infection.

  In order to solve this problem, the present inventors have conducted intensive studies. As a result, the inventors have found that a hematoxylin compound inhibits the interaction of Vpr protein and importin α to prevent nuclear translocation of Vpr protein, and further inhibits infection of HIV virus to macrophages, thereby completing the present invention.

That is, in the first aspect of the present invention, a human immunodeficiency virus infection inhibitor comprising a compound represented by the following formula (I), a pharmaceutically acceptable salt thereof, or a hydrate thereof as an active ingredient: An agent is provided.

  In another aspect of the present invention, there is provided an AIDS therapeutic agent or AIDS preventive agent comprising the inhibitor as an active ingredient.

The HIV infection inhibitor of the present invention can be used as a preventive or therapeutic agent for AIDS.
The HIV infection inhibitor of the present invention not only inhibits infection through inhibition of nuclear translocation of HIV Vpr protein, but also releases the G2 / M phase arrest of virus-producing cells by Vpr protein and reduces the virus production ability (He, J. et al., (1995) J. Virol. 69, 6705-6711) and by inhibiting apoptosis of virus-infected CD4-positive T cells, the host immune system can be treated with cytotoxic lymphocytes against infected cells. Inducing effects (Finkel, TH et al., (1995) Nature Med., 1, 129-134) can also be expected.

The compound useful as an inhibitor of the present invention includes a compound represented by the following formula (I), a pharmaceutically acceptable salt thereof, or a hydrate thereof.

This compound is a compound known as hematoxylin, but the use of hematoxylin is only known as a dyeing agent and the like, and its use as a pharmaceutical is not known.
Since this compound has an asymmetric carbon atom in the molecule, there are stereoisomers such as optical isomers or diastereomers. However, the scope of the present invention includes any stereoisomers in addition to pure stereoisomers. Body mixtures or racemates are included.

The compound represented by the formula (I) inhibits the interaction of “Vpr protein” and “importin α”, thereby preventing nuclear translocation of Vpr protein and inhibiting HIV virus infection. Therefore, the compound represented by the formula (I) can be used as an HIV infection inhibitor.
“Vpr protein” refers to the HIV-1 Vpr protein (eg, Ogawa K., et al., Journal of Virology, 1989, Vol. 63, No. 9, p. 4110-4114, and GenBank Accession No. M28355, SEQ ID NO: 2), but Vpr-like proteins (Vpr and / or Vpx) that are very well conserved in primate lentiviruses such as HIV-2 and SIV may be used.
Examples of “importin α” include the protein of SEQ ID NO: 4.

  The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. If the compounds according to the invention are acidic, their corresponding salts, for example alkali or alkaline earth metal salts such as calcium or magnesium salts, or ammonium salts, or methylamine, dimethylamine, trimethylamine, piperidine , Salts with organic bases such as morpholine or tris- (2-hydroxyethyl) amine. If the compounds according to the invention are sufficiently basic, their acid addition salts, for example inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, trifluoroacetic acid, citric acid or maleic acid, and And acid addition salts thereof.

  The compounds according to the invention may also be in the form of prodrugs. Various forms of prodrugs are known in the art, such as in vivo hydrolysable esters. In vivo hydrolysable derivatives include, in particular, pharmaceutically acceptable derivatives that can be oxidized or reduced in the human body to yield the parent compound, or esters that hydrolyze in the human body to yield the parent compound. Is included. Such esters can be identified by examining the body fluid of the test animal after intravenous administration of the compound under test, for example, to the test animal. In vivo hydrolysable esters suitable for hydroxy include acetyl.

(Pharmaceutical composition of the present invention)
When administered as a medicament, the HIV infection inhibitor of the present invention is administered to mammals including humans as a pharmaceutical composition as it is or together with a pharmaceutically acceptable non-toxic and inert carrier. Suitable carriers for oral administration may contain minor components such as buffers, fragrances and the like. The amount of carrier in the pharmaceutical composition is usually in the range of 5 to 95% of the total, but can vary greatly depending on the dosage form. Suitable carriers include sucrose, pectin, magnesium stearate, lactose, peanut oil, olive oil, water and the like.

  When the pharmaceutical composition of the present invention is administered to humans for the purpose of preventing AIDS-infected persons (asymptomatic patients) or treating patients after the onset of AIDS, powders, granules, tablets, capsules, pills It can be administered orally as a liquid or the like, or parenterally as an injection, a suppository, a transdermal absorption agent, an inhalant or the like. In addition, an effective amount of the inhibitor of the present invention is mixed with excipients, binders, wetting agents, disintegrants, lubricants, and other pharmaceutical additives suitable for the dosage form as necessary to obtain a pharmaceutical composition. It can be a thing. In the case of injection, it is sterilized with an appropriate carrier to prepare a preparation.

The pharmaceutical composition of the present invention can be used as a single therapeutic agent or in combination with other therapeutic agents. Examples of drugs that can be combined with the HIV infection inhibitor of the present invention include HIV reverse transcriptase inhibitors and protease inhibitors, immunostimulators such as various cytokines, and liposome preparations containing small interfering RNAs. included. The dosage of these pharmaceutical compositions varies depending on the disease state, administration route, patient age, or body weight, and is ultimately left to the judgment of a doctor. 1-100 mg / kg / day, preferably 1-20 mg / kg / day. When administered parenterally, usually 0.01-10 mg / kg / day, preferably 0.1-2 mg / kg / day Is administered. This may be administered once or divided into several times.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.

<Protein expression and purification>
Plasmids GST-N17C74-GFP-His ₆ and GST-Importin α-His ₆ , are polyhistidine (His ₆ ) in the sequence described in Journal of Virology, March 2005, p. 3557-3564, Vol. 79, No. 6. Built with tags.
GST-N17C74-GFP-His ₆ is a region formed by the 17th to 74th amino acid residues from the N-terminal end of Vpr, which is the minimal component domain of Vpr nuclear translocation including α-helix region that binds importin α ( JP 2006-067994 A: GST (glutathione-S-transferase) tag, GFP (Green fluorescence protein) tag, and His ₆ tag fusion protein are added to amino acid residues 17 to 74 of SEQ ID NO: 2. It is a plasmid for expression. GST-Importin α-His ₆ is a plasmid for expressing Importin α, a GST tag, and a polyhistidine tag fusion protein (GST-Impα-His ₆ ).

Recombinant Escherichia coli was obtained by transforming Escherichia coli with each of the above plasmids. Each fusion protein was expressed by culturing the resulting recombinant E. coli. The resulting fusion protein was purified using a GST column. GST-N17C74-GFP-His ₆ was cleaved with PreScission protease (Amersham Biosciences) to obtain N17C74-GFP-His ₆ (Vpr-GFP-His ₆ ). In addition, GST-N17C74-GFP-His ₆ was cleaved with TEV protease (Invitrogen) to obtain GST-N17C74. In addition, GST-Importin α-His ₆ was cleaved with PreScission protease to obtain Importin α-His ₆ .

Nuclear translocation of the Vpr protein is important for the preintegration complex containing the HIV-1 genome to cross the nuclear membrane of immune cells, and this nuclear translocation of the Vpr protein is mediated by importin α. Therefore, an ELISA assay and a GST pull-down assay were constructed as a system for measuring the interaction between the Vpr protein and importin α, and it was examined whether the compound inhibited the interaction between both proteins.
<GST pull-down assay>
N17C74-GFP-His ₆ is pre-glutathione-Sepharose 4FF in PBS / 1% BSA containing the test compound. GST-Impα-His ₆ adsorbed on beads (Amersham Pharmacia Biotech) was reacted at 4 ° C. for 1 hour. The beads were washed 6 times with PBS / 0.1% Triton X and then heated at 98 ° C. for 5 minutes to elute proteins bound to the beads. The eluted protein was separated by SDS-PAGE (15% polyacrylamide), transferred to a PVDF membrane (Immobilon; Millipore, Bedford, Mass.) And analyzed by immunoblot. The membrane was blocked with PBS / 5% skim milk for 1 hour and then reacted with an anti-GFP monoclonal antibody (MBL). After washing with PBS / 0.05% Tween 20, it was reacted with HRP-conjugated anti-mouse IgG. ^{Detection, SuperSignal TM West Pico Chemiluuminescent substrate (} Pierce,
Rockford, Ill).

<ELISA assay>
Anti-GST monoclonal antibody (SIGMA) dissolved in 50 mM NaHCO ₃ (pH 9.8) was added to each well of a collagen-coated microplate (NUNC) and coated at 4 ° C. for 6 hours. The wells were washed with PBS / 0.1% Tween20, blocked with PBS / 5% BSA (30 ° C, 2 hours), then GST or GST-N17C74 dissolved in PBS / 5% BSA was added at 0.5 μg / well. Incubated for 2 hours at 4 ° C. in the presence or absence of compound. After washing the wells, Importin α-His ₆ dissolved in PBS / 5% BSA was added at 1.0 μg / well and incubated at 4 ° C. for 2 hours. Thereafter, the solution was removed, and the plate was washed with PBS / 0.1% Tween 20, and then horseradish peroxidase-conjugated anti-His monoclonal antibody (Invitrogen) was added and incubated at 22 ° C. for 1 hour. The microplate was washed 3 times with PBS / 0.1% Tween 20, and 50 μl of tetramethyl benzidine (TMB) (Pierce) was added. After incubating at 37 ° C. for 30 minutes, a reaction stop solution was added, and the degree of binding between Vpr and Importin α was examined by measuring OD ₄₅₀ with an ELISA plate reader (WALAC).

<Nuclear transfer assay>
In vitro nuclear translocation assay was performed using digitonin-treated HeLa cells, Vpr-GFP-His ₆ and GST-Importin α-His ₆ . HeLa cells cultured to 80% confluence on a cover glass were transferred to a nuclear transfer analysis buffer (TB; 20 mM HEPES-KOH (pH 7.3), 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 2 mM EGTA , 1 mM dithiothreitol) and treated with 40 μg / ml digitonin (Fluka AG) at 4 ° C. for 5 minutes to make it permeable. Vpr-GFP-His ₆ and GST-Importin α-His ₆ were added to the nuclear transfer analysis buffer containing the test compound so that the final concentration was 1 μM, respectively, and 40 μl was added to the cells as a test sample. The nuclear transfer reaction was allowed to proceed at 28 ° C. for 15 minutes, and then the cells were washed 3 times with an ice-cooled nuclear transfer analysis buffer and fixed on ice in a nuclear transfer analysis buffer containing 4% formaldehyde for 30 minutes. About the sample produced by embedding with glycerol, the fluorescence of GFP was observed with the confocal laser scanning microscope (Radiance 2100, Bio-Rad).

<Infection assay>
According to the method described in Journal of Virology, May 2007, p. 5284-5293, Vol. 81, No. 10, monocytes were isolated from human peripheral blood mononuclear cells and differentiated into macrophages to prepare macrophages. .
After introducing the macrophage-directed molecular clone pYK-JRCSF into 293T cells using Fugene (Roche), the supernatant was collected 24 hours and 48 hours later, and the p24 ELISA method (HIV-1 p24 ^gag ELISA kit (RETRO TEC; ZeptoMetrix, ZeptoMetrix, NY)) and the amount of virus was quantified to obtain a stock virus.
For virus infection, macrophages cultured in a 24-well plate were inoculated with 2 ng of virus in the amount of p24 and infected at 37 ° C. for 3 hours. Thereafter, the plate was washed three times, transferred to a 24-well plate (Nunc), and cultured in RPMI1640 medium (sigma) supplemented with 10% calf fetal serum in the presence of the compound at 37 ° C. in a 5% CO ₂ atmosphere. After 8 days, the supernatant was collected and virus production was measured by the p24 ELISA method.

<Cytotoxicity assay>
To examine the cytotoxicity of the compounds, an MTT assay based on mitochondrial dehydrogenation activity was performed. Molt4T cell line was incubated at 37 ° C. for 2 hours in medium containing the compound in the presence of MTT at a final concentration of 0.75 mg / ml. Thereafter, the cells were lysed with a solution containing 90% isopropanol, 10% TritonX-100 and 1% HCl, and incubated at 37 ° C. for 24 hours. The produced MTT formazan was quantified using a microplate reader (Wallac ARVO SX 1420, Perkin-Elmer) (excitation wavelength 570 nm, emission wavelength 655 nm).

<Result>
When 2000 or more compounds were examined for binding inhibitory activity between Vpr and importin α by ELISA assay, 49 compounds were obtained as shown in Table 1. As a result of further evaluating the activity of inhibiting the interaction between Vpr protein and importin α by GST pull-down assay, 11 compounds were obtained. The nuclear translocation assay revealed that two compounds inhibit Vpr nuclear translocation.

ヘマトキシリン（H化合物） One of the two compounds was hematoxylin. The results of the hematoxylin ELISA assay and the GST pull-down assay are shown in FIG. FIG. 1 shows that hematoxylin inhibits the binding between Vpr and importin α.

Hematoxylin (H compound)

  As shown in FIG. 2, it was confirmed that hematoxylin inhibits nuclear translocation of Vpr protein.

  Moreover, as a result of investigating whether or not hematoxylin actually inhibits HIV infection by an infection assay, it was confirmed that hematoxylin inhibits HIV infection of macrophages as shown in FIG. Further, as shown in FIG. 4, it was shown that the cytotoxicity is low in the Molt4T cell line.

(A) A diagram (photograph) showing the results of an ELISA assay using GST-N17C74 and Importin α-His ₆ . Impβ is a positive control importin β, and H is hematoxylin. (B) GST-Impα-His 6 and Vpr-GFP-His shows the results of GST pull-down assay using FIG (photograph). The GFP lane shows GFP reacted with GST-Impα-His ₆ . As a control, only the solvent was added to the reaction solution. As a result, the compound (hematoxylin) inhibited the binding of Impα and Vpr in a concentration-dependent manner (1 μM, 10 μM, 100 μM). The figure (photograph) which shows the result of a nuclear-transfer assay using digitonin treatment HeLa cell. The compound (hematoxylin) inhibited Vpr nuclear translocation in a concentration-dependent manner (1 μM, 10 μM, 100 μM). The figure which shows the result of the infection assay to a macrophage. The horizontal axis indicates the compound concentration (μM). ● and ■ are the results of hematoxylin, and the origin of the macrophages used in the assay is different. The figure which shows the result of a cytotoxicity assay. The horizontal axis indicates the compound concentration (μM).

Claims (2)

A human immunodeficiency virus infection inhibitor comprising a compound represented by the following formula (I), a pharmaceutically acceptable salt thereof, or a hydrate thereof as an active ingredient:

A medicament for treating or preventing AIDS, comprising the inhibitor according to claim 1 as an active ingredient.

Images