JP2006321759A - INHIBITOR OF Vpr PROTEIN FUNCTION AND REMEDY FOR AIDS CONTAINING THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To discover a remedy for AIDS based on a new mechanism of action, by searching such a compound as to inhibit cytostatic activity of Vpr protein. <P>SOLUTION: An inhibitor of a human immunodeficiency virus Vpr protein function contains a compound expressed by general formula (I) (R<SP>1</SP>and R<SP>2</SP>are each H or -OR<SP>3</SP>, wherein R<SP>3</SP>is an alkyl which may be substituted or the like), a pharmaceutically acceptable salt thereof, or a hydrate thereof as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an inhibitor of Vpr protein function, a new type of AIDS preventive and therapeutic agent containing the inhibitor, and a screening method thereof.

  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. Only enzyme inhibitors and protease inhibitors such as indinavir (crixiban) and saquinavir. Although these drugs are effective for AIDS treatment, HIV-1 is easily mutated, and one problem is that drug-resistant strains appear in the course of treatment. The therapy using a combination of various drugs has an effect of suppressing the appearance of drug-resistant strains. However, since currently available therapeutic agents are limited to those targeting HIV reverse transcriptase and protease as described above, there are limits 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 connection with HIV replication, eg, activating transcription of various retroviral LTRs, including HIV, or Vpr is a G ₂ / M cell cycle. The cell cycle is disturbed by accumulating cells stopped in the phase (see, for example, Non-Patent Documents 1 and 2). In cells arrested in the G ₂ / M phase due to the function of Vpr, virus replication is increased 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, the present inventors have already reported that a compound isolated from a fermentation liquid of a certain filamentous fungus selectively suppresses the proliferation of vascular endothelial cells (for example, see Patent Document 1). Angiogenesis is becoming an important physiological phenomenon in the growth and metastasis of various advanced solid cancers. Among them, compounds that selectively inhibit proliferation of vascular endothelial cells are expected to be applied to angiogenesis inhibitors, antitumor agents, metastasis inhibitors, antirheumatic arthritis agents, and the like. However, the details of the mechanism by which this compound selectively inhibits the proliferation of vascular endothelial cells is not clear, and the effect on the proliferation of AIDS virus is not known.

Japanese Patent Laid-Open No. 2004-262881 Emerman, M. (1996) Curr. Biol. Vol. 6, pp. 1096-1103. Shimura, M., Zhou, Y., Asada, Y., Yoshikawa, T., Hatake, K., Takaku, F., and Ishizaka, Y., (1999) Biochemical and Biophysical Research Communication Vol. 261, No. 2, pp.308-316. Hoch J. et al., (1995) J. Virol. Vol. 69, pp. 4807-4813. Macreadie, I.G., Castelli, L.A., Hewish, D.R., Kirkpatrick, A., Ward, A.C. and Azad, A.A., (1995) Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 2770-2774.

  An object of the present invention is to find an AIDS therapeutic agent based on a novel mechanism of action by searching for a compound that inhibits the cell growth arresting ability of Vpr protein known to be important for the proliferation of HIV. And

  In order to solve such a problem, the present inventors constructed a new screening system using the fact that Vpr protein stops the growth of budding yeast, and uses this in a certain filamentous fungus culture medium. The present invention has been completed by finding a substance that inhibits the function of and isolating and identifying the substance.

That is, in the first aspect of the present invention, a human immunodeficiency virus Vpr comprising a compound represented by the following general formula (I), a pharmaceutically acceptable salt thereof, or a hydrate thereof as an active ingredient: Inhibitors of protein function are provided.

However, R < ¹ > and R < ² > shows a hydrogen atom or -OR < ³ > mutually independently,
R ³ represents an alkyl group, an alkenyl group, an alkanoyl group, an alkenoyl group, a carbamoyl group, or a substituent represented by the following formula (II), each of which may have a substituent,
—C (═O) — (CH═CH) _n —R ⁴ (II)
However, n is an integer of 2 to 5, R ⁴ represents a hydrogen atom, a methyl group, or a carboxyl group, or R ¹ and R ² may be combined to form a carbonyl group.

In a preferred embodiment, the compound is a compound represented by the following formula (III) or (IV), a geometric isomer thereof, or an optical isomer thereof.

  In another aspect of the present invention, there is provided an AIDS therapeutic agent comprising the inhibitor as an active ingredient in the form of a single agent or a mixed agent.

  Still another aspect of the present invention is characterized in that a compound showing a growth inhibitory effect on human umbilical vein vascular endothelial cells or a derivative thereof is used as a candidate compound, and it is measured whether or not it inhibits the Vpr protein function of HIV-1. Methods for screening for AIDS therapeutics are provided. The candidate compound is preferably an O-substituted fumagillol derivative.

  Since the Vpr function inhibitor of the present invention effectively inhibits the function of the Vpr protein required for HIV infection and proliferation and inhibits the destruction of various cells based on HIV infection, the prevention or treatment of the onset of AIDS It can be used as a medicine. In addition, since the screening method of the present invention can easily and efficiently search for whether or not the function of the Vpr protein is inhibited using the growth of budding yeast as an index, an AIDS therapeutic drug can be selected from known compounds. It is useful as a method of finding out. In particular, by using as a candidate compound a group of compounds that show the growth inhibitory effect of human umbilical vein endothelial cells, it is possible to find a medicinal effect as an anti-AIDS drug among compounds developed as angiogenesis inhibitors and antitumor agents. It is extremely useful as a system that can rapidly screen for a highly safe AIDS therapeutic agent that is capable of low toxicity.

(Inhibitor of Vpr protein function)
As used herein, the term “inhibitor” includes any suitable molecule, compound, protein or fragment thereof, nucleic acid, or formulation that can suppress the function (activity) of the Vpr protein. Here, “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. The term “Vpr protein function” includes many biological functions of Vpr protein, such as weak activation of several viral promoters (Cohen, EA et al., (1990) J. Acquir. Immune Defic . Syndr. 3, 11-18), serves to stop the host cells in G _{2 /} M phases of the cell cycle (He, J. et al., (1995) J. Virol. 69, 6705-6711), infected cells Action to suppress the proliferation of cells and induce apoptosis (Finkel, TH et al., (1995) Nature Med., 1, 129-134), promotion of HIV-1 preincorporation complex (PIC) translocation to the nucleus ( Heinzinger, NK et al., Proc. Natl. Acad. Sci. USA. (1994) 91, 7311-7315; Subbramanian, RA et al., (1998) J. Exp. Med. 187, 1103-1111), and Interactions with various cytoplasmic factors such as glucocorticoid receptors (Kino, T. et al., (1999) J. Exp. Med. 189, 51-61).

  In the present specification, the “inhibitor of Vpr protein function” may also be referred to as “Vpr inhibitor”.

Compounds useful as inhibitors of the present invention include, but are not limited to, compounds represented by general formula (I) below, pharmaceutically acceptable salts, or hydrates thereof.

However, R < ¹ > and R < ² > shows a hydrogen atom or -OR < ³ > mutually independently,
R ³ represents an alkyl group, an alkenyl group, an alkanoyl group, an alkenoyl group, a carbamoyl group, or a substituent represented by the following formula (II), each of which may have a substituent,
—C (═O) — (CH═CH) _n —R ⁴ (II)
However, n is an integer of 2 to 5, R ⁴ represents a hydrogen atom, a methyl group, or a carboxyl group, or R ¹ and R ² may be combined to form a carbonyl group.

  In this specification, the term “alkyl group” means methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl and the like, unless the chain length is particularly limited. These include both straight and branched chain substituents of 1 to 10 carbon atoms including but not limited to these.

  The term “alkenyl group” includes, but is not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like, unless the chain length is limited. Means a linear or branched substituent of 2 to 10 carbon atoms having at least one double bond between two carbon atoms in the chain.

  “Alkanoyl” is C (O) -alkyl, where alkyl is as defined above, and “alkenoyl” is C (O) -alkenyl, where alkenyl is as defined above.

  Specific compounds of general formula (I) include, for example, fumagillin (compound represented by formula (IV)), 6-O-octatrienoyl fumagillol, 6-oxo-6-deoxy fumagillol, 6- Examples include, but are not limited to, O-chloroacetylcarbamoyl fumagillol. Since these compounds have asymmetric carbon atoms at a plurality of positions in the molecule, there are stereoisomers such as optical isomers or diastereomers. However, the scope of the present invention includes pure stereoisomers. In addition, a mixture of arbitrary stereoisomers or a racemate is included. These compounds are disclosed in, for example, JP-T-2003-502324, JP-A-6-220034, or Asami, Y. et al., (2004) Tetrahedron, Vol. 60, pp.7085-7091, or a method according to them. Can be manufactured according to.

  The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When 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 hydrolyzable 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 hydrolyzable esters suitable for hydroxy include acetyl, carboxyl includes, for example, alkyl esters, dialkylaminoalkoxy esters, alkoxymethyl esters such as methoxymethyl, alkanoyloxymethyl such as pivaloyloxymethyl, etc. Esters, cycloalkoxycarbonyloxyalkyl esters such as 1-cyclohexylcarbonyloxyethyl, 1,3-dioxolan-2-ylmethyl esters such as 5-methyl-1,3-dioxolan-2-ylmethyl, and 1-methoxycarbonyloxy Alkoxycarbonyloxyethyl esters such as ethyl are included.

Preferred compounds as the inhibitor of the present invention include compounds represented by the following formula (III) or formula (IV), geometric isomers thereof, or optical isomers thereof.

  The compound represented by formula (III) or formula (IV) has an asymmetric carbon, and there exist stereoisomers such as optical isomers or diastereomers based on the asymmetric carbon. In addition to the stereoisomers in pure form, any stereoisomer mixture or racemate is included. In addition, the above compound has a plurality of olefinic double bonds, and geometric isomers based on the respective double bonds may exist. Body mixtures are also encompassed within the scope of the present invention. Among the compounds represented by the formula (III), a particularly preferable compound is called “RK-95113”, a method of separating and collecting from fermentation products of microorganisms (extraction method), and chemical synthesis (synthesis) using them as starting materials. Method). In the extraction method, a compound capable of producing compound (III) is cultured under the medium composition and medium conditions used for normal fermentation production, and compound (III) is produced and accumulated in the medium, and then separated and collected. (III) is isolated.

  Examples of the microorganism capable of producing the compound (III) according to the present invention include the filamentous fungus RK95-113. The microorganism was deposited under the accession number FERMP-19233 on February 26, 2003 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Tokuba 1-1-1, Tsukuba, Ibaraki). Has been.

  As a production medium for the compound (III) of the present invention, either a synthetic medium or a natural medium can be suitably used as long as it appropriately contains a carbon source, a nitrogen source and an inorganic salt. If necessary, vitamins or other nutrient substances may be added as appropriate. Examples of carbon sources include sugars such as glucose, maltose, fructose, sucrose and starch, alcohols such as glycerol and mannitol, amino acids such as glycine, alanine and asparagine, and fats and oils such as soybean oil and olive oil. One kind or two or more kinds may be appropriately selected and used from various carbon sources in consideration of assimilability of microorganisms. Nitrogen sources include soy flour, corn steep liquor, beef extract, peptone, yeast extract, amino acid mixture, organic nitrogen-containing compounds such as fish meal, or inorganic nitrogen compounds such as ammonium salts and nitrates, taking into consideration the assimilability of microorganisms Thus, one or more kinds may be appropriately selected and used. As the inorganic salt, for example, calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, copper sulfate, manganese chloride, zinc sulfate, cobalt chloride, and various phosphates may be added as necessary. Further, an antifoaming agent such as vegetable fat, polypropylene alcohol and the like can be added as necessary.

  The culture temperature can be appropriately changed within the range in which microorganisms grow and produce compound (III), but is preferably 10-32 ° C, more preferably 20-28 ° C. The initial pH is desirably around 6-8, and the culture time is usually about 4 days to several weeks, but when the production amount of the compound (III) of the present invention reaches a collectable amount, preferably when it reaches the maximum. In this case, the culture is terminated. Any culture method can be suitably used as long as it is a commonly used method such as solid phase culture or aeration and agitation culture.

  In order to separate and isolate the compound (III) from the culture broth, it is generally possible to appropriately utilize means commonly used for collecting microbial metabolites. For example, various ion exchange resins, nonionic adsorption resins, gel filtration chromatography, chromatography with an adsorbent such as activated carbon, alumina, silica gel, or high performance liquid chromatography, crystallization, concentration under reduced pressure, or lyophilization The means can be used alone or in appropriate combination or repeatedly.

(Inhibition of Vpr protein function and screening for AIDS drugs)
Whether or not the Vpr protein function is inhibited can be determined by examining the influence of the compound of the present invention in any system capable of measuring the function of the Vpr protein. For example, adding the compound of the present invention to a mammalian cell expressing the vpr gene and culturing, and observing whether the function of the Vpr protein is inhibited by cell cycle analysis, cell morphological observation, etc. Can do. Alternatively, a reporter gene may be introduced downstream of a promoter sequence that is transcriptionally controlled by the Vpr protein, and the activity of the reporter gene in animal cells co-expressed with the vpr gene may be measured. As described in Examples described later, a series of compounds of the present invention has been found to inhibit the cell growth inhibitory function of Vpr protein in a screening system using yeast.

  Such an inhibitor of Vpr protein function is considered to have the following effects on HIV infection, proliferation, and AIDS development. 1) Release the G2 / M phase arrest of virus-producing cells by the Vpr protein and reduce the virus-producing ability. 2) Inducing cytotoxic lymphocytes against infected cells in the host immune system by inhibiting apoptosis of virus-infected CD4-positive T cells. 3) Inhibits HIV-1 infection and proliferation by inhibiting viral genome transfer into the nucleus of non-dividing cells such as macrophages. Furthermore, disruption of the cell cycle by Vpr promotes canceration of normal cells, and it may lead to the development of AIDS-related tumors such as Kaposi's sarcoma with a decrease in immune function, thus inhibiting the function of Vpr protein. By doing so, it is thought that not only the onset of AIDS but also its progression can be suppressed.

  Therefore, in a different aspect of the present invention, there is provided a method for screening for an AIDS therapeutic agent using the fact that Vpr protein stops the growth of budding yeast. It has been reported that when the Vpr protein is expressed in yeast cells, yeast cell growth is stopped (see Non-Patent Document 4 above). The present inventors have applied this system to construct a novel Vpr inhibitor screening system. That is, a paper disk containing a candidate compound is placed on an agar medium containing yeast that has stopped growing due to Vpr expression, and the yeast grows when the Vpr function is inhibited by the candidate compound exuded from the paper tissue. To search for Vpr inhibitors.

  Candidate compounds can be natural products or extracts thereof, secondary metabolites of microorganisms, synthetic compounds, etc., which are prepared using solutions dissolved at various concentrations in water or culture solutions. . For compounds in which the growth of yeast has been observed, a solution diluted in stages can be prepared, and the minimum effective concentration can be measured to provide an indicator of activity.

  The compound of the present invention was found from a secondary metabolite of a microorganism using such a screening system, and the present inventors have already reported that it has an angiogenesis inhibitory action. In particular, the compounds of formulas (III) and (IV) inhibit the growth of normal human umbilical vein endothelial cells HUVECs by 50% at a concentration of 0.1 ng / ml, but human normal lung fibroblasts WI− It has been found that a concentration of 10 ng / ml or higher is required to inhibit the growth of 38 by 50%. The relationship between the selective growth suppression of vascular endothelial cells and the suppression of Vpr protein function is not clear, but may be based on a common mechanism such as, for example, inhibiting interaction with a specific protein. There is also.

  Therefore, in one embodiment of the present invention, a compound showing a growth inhibitory effect of human umbilical vein vascular endothelial cells or a derivative thereof is used as a candidate compound to determine whether or not it inhibits the Vpr protein function of HIV-1. A screening method for a featured AIDS therapeutic agent is provided. The skeleton common to the compounds of the present invention may provide a molecular structure common to the selective growth inhibition of vascular endothelial cells and the inhibition of Vpr protein function. However, since the interaction between the compound of the present invention and the protein may be influenced by a minute change in the three-dimensional structure on the protein side, a strict correlation is not necessarily established. Therefore, even a compound having a relatively small ability to suppress the proliferation of vascular endothelial cells may strongly inhibit the Vpr protein function. Therefore, a compound similar to the compound of the present invention, which suppresses the proliferation of vascular endothelial cells, may contain a potent inhibitor of Vpr protein function. Such compounds include O-substituted fumagillol derivatives. Until now, the physiological activity of many O-substituted fumagillol derivatives has been reported, but the effect on the Vpr protein is not yet known. Therefore, there is a possibility that compounds useful as anti-AIDS drugs can be discovered by examining whether these compounds inhibit Vpr protein function by the method of the present invention. In particular, it is often experienced that some compounds that have dropped out of their original purpose due to side effects such as toxicity and absorbability during drug development show significant effects for other purposes. Is Rukoto.

(Pharmaceutical composition of the present invention)
When administered as a pharmaceutical, the Vpr 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, suppository, transdermal absorption agent, 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. Drugs that can be combined with the Vpr inhibitor of the present invention include HIV reverse transcriptase inhibitors and protease inhibitors, immunostimulators such as various cytokines, and liposome preparations containing small interfering RNA. It is. 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.

[Screening of inhibitors of Vpr function using the applied yeast]
A screening system for inhibitors of Vpr function was constructed using a system (Macreadie et al.) That expresses Vpr protein in budding yeast to stop yeast growth. That is, from the plasmid pMT-FVpr (see Watanabe et al. Exp. Cell Res. 258, 261-269), the Vpr gene with the N-terminal FLAG tag was excised, and the budding yeast expression vector pYEX-BX (Clontech, catalog #). 6199-1) was inserted downstream of the copper ion (Cu ²⁺ ) inducible CUP1 promoter (derived from the yeast metallothionein gene) to construct an expression vector in yeast. This expression vector was introduced into the budding yeast strain MLC30 (see Miyamoto et al., J. Biol. Chem., 2002, 277, 28810-28814).

  This yeast was mixed and cultured in a synthetic minimum agar medium containing 0.5 mM copper sulfate and 0.001% SDS but not leucine or uracil. Under these conditions, the yeast growth is stopped by the expression of the Vpr protein in the yeast cells. FIG. 1 shows a paper in which an agar medium (10) containing yeast whose growth has been stopped by Vpr expression is prepared in a rectangular petri dish, and secondary metabolites of microorganisms, compounds derived from a synthetic compound library, and the like are contained on the agar medium. It is the figure which showed typically the system which mounts a disc (filter paper about 6 mm in diameter) (20), and screens a Vpr function inhibitor. When this petri dish was cultured at 30 ° C. for 4 days, the growth of yeast was observed around the paper disk containing the inhibitor of Vpr function (30). FIG. 2 shows a photograph of yeast grown around filter paper containing 10 μl of a purified methanol solution (2 mg / ml) of fumagillin (Sigma, catalog number F6771). The diameter of the circle formed by yeast growth was about 1.3 cm.

[Verification of HIV-1 infection suppression effect by Vpr inhibitor]
A luciferase reporter virus prepared according to the method of Connor et al. (Connor et al, (1995) Virology, 206, 935-944) is used to quantitatively detect HIV infection in various cells and to inhibit Vpr inhibitors The HIV-1 infection suppression effect was verified. That is, the plasmid pNL-Luc-E ^- R ⁺ is an (env ⁻ ) retrovirus vector having a mutation in the envelope gene so that it cannot self-replicate to the infectious provirus NL4-3 of vpr ⁺ , and is contained in the nef gene. The firefly luciferase gene is inserted. The vpr gene of plasmid frameshift mutations introduced pNL-Luc-E ^- R ^- is Vpr protein can not be expressed. The ^pNL-Luc-E - ^{R +} or ^{pNL-Luc-E - R -} , and 293 cells were co-transfected with pSV-A-MLV-env is the envelope protein expression vectors wide host resistance, luciferase reporter Virus was made. For virus infection, various cells cultured in a 24-well incubator were inoculated with 10 ng of virus as a p24 amount. After overnight culture in Dulbecco's Modified Eagle (DMEM) medium supplemented with 10% calf fetal serum at 37 ° C. in a 5% CO ₂ atmosphere, the cells were washed twice, followed by 1.5 ml of RPMI / 10% FCS. Or 10 U / ml IL-2 supplemented medium. After a predetermined time, the cells were lysed in 200 μl of lysis buffer (Promega) and stored at −70 ° C. The luciferase activity in each 20 μl of lysate was measured with a Lumat LB9501 luminometer using a commercially available reagent (Promega).

As shown in FIGS. 3 to 5, vpr ⁺ virus expresses luciferase when infecting cells, and its activity can be quantified by measuring its activity, but Vpr ⁻ mutant virus can be infected under the same conditions. However, luciferase activity can hardly be detected even if it is used, and it is understood that Vpr is necessary for the establishment of HIV-1 infection.

Human peripheral blood-derived macrophages were infected with virus (Vpr ⁺ or Vpr ⁻ , 10 ng as the amount of p24), and fumagillin solution was added 12 hours later. The results of measuring luciferase activity after 7 days are shown in FIG. About 50% inhibition was observed in cells to which fumagillin was added at 1 μg / ml or 10 μg / ml as compared to control cells without any drug. The fumagillin solution was obtained by purifying an ethyl acetate extract from a filamentous fungus culture with silica gel chromatography and HPLC and then recrystallizing (the chemical structure was confirmed by NMR and mass spectrometry) or Sigma (catalog number F6771) from methanol or DMSO. And dissolved in 2 mg / ml.

  Next, human myelomonocytic cell line U937 cells were infected with the virus by the same method, fumagillin solution was added after 6 days (10 μg / ml, 1 μg / ml, 100 ng / ml), and luciferase activity was measured after 3 days. (See FIG. 4). As shown in FIG. 4, luciferase activity, ie, viral infection, was inhibited by about 70% at any concentration.

  Further, U937 cells were infected with the virus, and immediately the fumagillin solution was added (10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml), and luciferase activity was measured after 9 days (see FIG. 5). As shown in FIG. 5, when fumagillin was added at a concentration of 10 μg / ml, luciferase activity, ie, viral infection, was inhibited by about 85%.

  From the above results, it was confirmed that the Vpr inhibitor of the present invention significantly inhibits HIV-1 infection.

A method for screening a candidate compound for a Vpr inhibitor on an agar medium containing yeast is schematically shown. FIG. 2 shows yeast growing around filter paper containing 10 μl of fumagillin solution (2 mg / ml) on the agar medium shown in FIG. It is the result of measuring luciferase activity 7 days after infecting two kinds of viruses in macrophages and adding or not adding fumagillin solution. It is the result of infecting U937 cells with virus, adding fumagillin solutions of various concentrations after 6 days, and measuring luciferase activity after 3 days. It is the result of measuring luciferase activity 3 days after infecting U937 cells with virus and immediately adding various concentrations of fumagillin solution.

Explanation of symbols

10 Agar medium 20 Filter paper 30 Yeast production circle

Claims (7)

An inhibitor of human immunodeficiency virus Vpr protein function comprising a compound represented by the following general formula (I), a pharmaceutically acceptable salt thereof, or a hydrate thereof as an active ingredient:
However, R < ¹ > and R < ² > shows a hydrogen atom or -OR < ³ > mutually independently,
R ³ represents an alkyl group, an alkenyl group, an alkanoyl group, an alkenoyl group, a carbamoyl group, or a substituent represented by the following formula (II), each of which may have a substituent,
—C (═O) — (CH═CH) _n —R ⁴ (II)
However, n is an integer from 2 to 5, ^{R 4} represents a hydrogen atom, a methyl group, or carboxyl group,
Alternatively, R ¹ and R ² may be combined to form a carbonyl group. The inhibitor according to claim 1, wherein the compound is a compound represented by the following formula (III), a geometric isomer thereof, or an optical isomer thereof.
The inhibitor according to claim 1, wherein the compound is a compound represented by the following formula (IV), a geometric isomer thereof, or an optical isomer thereof.
  The inhibitor according to any one of claims 1 to 3, wherein the Vpr protein function is a cell growth arresting ability.   A therapeutic agent for AIDS comprising the inhibitor according to any one of claims 1 to 4 as an active ingredient in the form of a single agent or a mixed agent.   A method for screening an AIDS therapeutic agent, comprising measuring a compound that exhibits an inhibitory effect on proliferation of human umbilical vein endothelial cells or a derivative thereof as a candidate compound, and inhibiting whether or not the Vpr protein function of HIV-1 is inhibited.   The method according to claim 6, wherein the candidate compound is an O-substituted fumagillol derivative.