JP2005120002A - Inhibitor against specific structure formation of amyloid precursor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an inhibitor against structural change of prion useful as a new therapeutic agent for prion disease. <P>SOLUTION: The inhibitor against structural change of prion protein comprises 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide or its derivative as an active ingredient. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to the treatment and prevention of prion diseases, and relates to compounds that suppress the conversion of normal prions to infectious prions.

Prion is sheep scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, GSS, FFI, Kuru (SB Prusiner, Science, 216, 136-144 (1982), SB Prusiner, Science, 252, 1515-1522 (1991) SB Prusiner, Prions. Proc. Natl. Acad. Sci. USA, 95, 13363-13383 (1998)), mutant Jacob disease (G. Chazot et al., Lancet, 347, 1181-1181 (1996), RG Causes neurodegenerative diseases such as Will et al., Lancet, 347, 921-925 (1996)). These diseases are called prion diseases. The cause of these diseases is the abnormality of prion protein, that is, the structural conversion from normal prion (PrP ^C ) to infectious prion (PrP ^Sc ) via amyloid precursor special structure (PrP ^* ) (SB Prusiner, Science, 216, 136-144 (1982), SB Prusiner, Science, 252, 1515-1522 (1991), K. Kuwata, et al., Biochemistry 41, 12277-12283 (2002) ).

  Until now, various attempts have been made to inactivate prions or attenuate infectivity, but prion resistance to conventional inactivation and infectivity attenuation methods by radiation, boiling, chemicals, etc. is strong, not necessarily The effect was not improved.

  When the infectious prion enters the human body, it binds with the normal prion in the body, and the normal prion is successively converted into the infectious prion. Although this conversion process has not been clarified, it is considered possible to develop anti-prion drugs based on the normal structure (V. Perrier et al., Proc. Natl. Acad. Sci. USA 97, 6073-6078 (2000)).

To date, mice (R. Riek et al., Nature, 382, 180-182 (1996)), hamsters (TL James et al., Proc. Natl. Acad. Sci. USA 94, 10086-10091 (1997)) Cattle (F. Lopez Garcia, R. Zahn, R. Riek, K. Wuthrick, Proc. Natl. Acad. Sci. USA 97, 8334-8339 (2000)), human (L. Calzolai et al., Proc. Natl. Acad. Sci. USA 97, 8340-8345 (2000)), three-dimensional structure determination by NMR is performed. These structures are quite similar, and from 128 to 231 are composed of a solid structure consisting of an α helix and an incomplete soft β sheet. From the N-end to No. 113, no structure is formed, and from No. 113 to No. 128 a hydrophobic cluster is formed, forming a plurality of structures (H. Liu et al., Biochemistry 38, 5362- 5377 (1999)). So far, prion intermediates have been observed (H. Zhang et al., Biochemistry 36, 3543-53 (1997), S. Hornemann, RA Glockshuber, Proc. Natl. Acad. Sci. USA 95, 6010-6014. (1998), AC Apetri, WK Surewicz, Biol. Chem. 277, 44589-44592 (2002)). In recent years, a prion folding intermediate, PrP ^*, has been identified by high-pressure NMR (K. Kuwata, et al., Biochemistry 41, 12277-12283 (2002)), and its structural features have been elucidated. In PrP ^* , helices B and C are denatured, but helix A is retained. PrP ^* is known to be present at 1% under physiological conditions (K. Kuwata, et al., Biochemistry 41, 12277-12283 (2002)).

V. Perrier et al., Proc. Natl. Acad. Sci. U. S. A. 97, 6073-6078 (2000) R. Riek et al., Nature, 382, 180-182 (1996) T. L. James et al., Proc. Natl. Acad. Sci. U. S. A. 94, 10086-10091 (1997) F. Lopez Garcia, R. Zahn, R. Riek, K. Wuthrick, Proc. Natl. Acad. Sci. U. S. A. 97, 8334-8339 (2000) L. Calzolai et al., Proc. Natl. Acad. Sci. U. S. A. 97, 8340-8345 (2000) H. Liu et al., Biochemistry 38, 5362-5377 (1999) H. Zhang et al., Biochemistry 36, 3543-53 (1997) S. Hornemann, R. A. Glockshuber, Proc. Natl. Acad. Sci. U. S. A. 95, 6010-6014 (1998) A. C. Apetri, W. K. Surewicz, Biol. Chem. 277, 44589-44592 (2002) K. Kuwata, et al., Biochemistry 41, 12277-12283 (2002)

  The present invention provides a prion structure conversion inhibitor that can be used as a novel prion disease therapeutic agent.

As mentioned above, prion intermediates have been observed (H. Zhang et al., Biochemistry 36, 3543-53 (1997), S. Hornemann, RA Glockshuber, Proc. Natl. Acad. Sci. USA 95, 6010. -6014 (1998), AC Apetri, WK Surewicz, Biol. Chem. 277, 44589-44592 (2002)). In recent years, a prion folding intermediate, PrP ^*, has been identified by high-pressure NMR (K. Kuwata, et al., Biochemistry 41, 12277-12283 (2002)), and its structural features have been elucidated. In PrP ^* , helices B and C are denatured, but helix A is retained. PrP ^* is known to be present at 1% under physiological conditions (K. Kuwata, et al., Biochemistry 41, 12277-12283 (2002)). The present inventors have shown that if PrP ^* is an essential intermediate when PrP ^C is converted to PrP ^Sc , an agent that suppresses PrP ^C fluctuation and strongly suppresses PrP ^* production will inhibit PrP ^Sc production. We thought that it would be suppressed and conducted an intensive study.

The inventors focused on the binding pocket between the A-S2 loop and the C-terminal side of helix B among the binding pockets present in the normal prion. First, the effects of many compounds were examined by computer docking simulation. As a result, we named 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino) -benzyl] -phenyl], which we named GN8420
The inventors have found that -acetamide suppresses the structural conversion of normal prions to infectious prions, and have completed the present invention.

（ここで、R₁、R₂、R₃およびR₄は独立にH、または炭化水素基である）
で表される[１]または[２]の化合物、
[４] 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]
-acetamideまたはその誘導体である[３]の化合物、
[５] [１]から[４]のいずれかの化合物を有効成分として含むプリオンタンパク質構造変換抑制剤、
[６] [１]から[４]のいずれかの化合物を有効成分として含むプリオン病の予防または治療剤、
[７] プリオン病が、羊のスクレイピー、ウシ海綿状脳症、クロイツフェルト・ヤコブ病、GSS、FFI、クールーおよび変異型ヤコブ病からなる群から選択される[６]のプリオン病の予防または治療剤、
[８] [１]から[４]のいずれかの化合物を調製し、該化合物を正常型プリオンタンパク質に接触させることを含む、正常型プリオンが感染型プリオンに変換されるのを抑制する方法、
[９] ドッキングシミュレーション用ソフトウェアを動作可能に有するコンピューターであって、正常型プリオンの３次元立体構造を記憶媒体に格納したコンピューターに候補化合物の３次元立体構造を入力する工程、コンピューター上で正常型プリオンと前期候補化合物のドッキングシミュレーションを行う工程を含む、プリオンタンパク質の構造変換を抑制し得る化合物のスクリーニング方法、ならびに
[１０] 候補化合物が、2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)
-benzyl]-phenyl]-acetamideの誘導体である、[９]のプリオンタンパク質の構造変換を抑制し得る化合物のスクリーニング方法。 That is, the present invention is as follows.
[1] Inhibits the conversion of normal prions to infectious prions by binding to the binding pocket of normal prion protein (PrP ^C ) and suppressing the generation of fielding intermediate prion protein (PrP ^* ) Compound,
[2] The compound according to [1], wherein a binding pocket is present between the A-S2 loop of normal prion protein (PrP ^C ) and the C-terminal side of helix B.
[3] General formula (I)

(Where R ₁ , R ₂ , R ₃ and R ₄ are independently H or a hydrocarbon group)
[1] or [2] compound represented by:
[4] 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino) -benzyl] -phenyl]
-acetamide or its derivative [3] compound,
[5] A prion protein structure conversion inhibitor comprising any one of the compounds of [1] to [4] as an active ingredient,
[6] A prophylactic or therapeutic agent for prion disease comprising the compound according to any one of [1] to [4] as an active ingredient,
[7] The agent for preventing or treating prion disease according to [6], wherein the prion disease is selected from the group consisting of sheep scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, GSS, FFI, Kuru, and mutant Jacob disease ,
[8] A method for suppressing the conversion of normal prion to infectious prion, comprising preparing the compound of any one of [1] to [4] and contacting the compound with normal prion protein;
[9] A computer having docking simulation software operable, the step of inputting a three-dimensional structure of a candidate compound into a computer storing a three-dimensional structure of a normal prion in a storage medium, a normal type on the computer A method for screening a compound capable of suppressing the structural transformation of a prion protein, including a step of docking simulation of a prion and a candidate compound in the previous period; and
[10] The candidate compound is 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino)
-benzyl] -phenyl] -acetamide, a method for screening a compound capable of suppressing the structural conversion of the prion protein of [9].

As shown in the examples, GN8420 (2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl
-acetylamino) -benzyl] -phenyl] -acetamide) binds to normal prion PrP ^C, inhibited structural conversion to abnormal prion PrP ^Sc. Therefore, the composition containing GN8420 or a derivative thereof is useful as a preventive or therapeutic agent for prion diseases.

Hereinafter, the present invention will be described in detail.
The present invention is a compound that suppresses the structural conversion of prion protein from normal prion (PrP ^C ) to infectious prion (PrP ^Sc ). The compounds of the present invention is to suppress the fluctuation of the structure of PrP ^C by binding to the binding pocket of PrP ^C, PrP ^C is prevented from being converted into PrP ^Sc. That is, according to the present invention, normal prion is converted into infectious prion by binding to the binding pocket of normal prion protein (PrP ^C ) and suppressing the production of folding intermediate prion protein (PrP ^* ). It is a compound that can bind to the binding pocket existing between the A-S2 loop of normal prion protein (PrP ^C ) and the C-terminal side of helix B.

  As an example of the compound of the present invention, a compound represented by the following formula: 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino) -benzyl] -phenyl]- acetamide and its derivatives.

  The derivative of the compound represented by the above formula refers to a compound in which a substituent is introduced into the basic skeleton represented by the above formula, and examples thereof include a compound in which a hydrocarbon or other substituent is further introduced into the pyrrolidine ring.

で表される化合物が挙げられる。 As an example of a derivative, the general formula (I)

The compound represented by these is mentioned.

Here, R ₁ , R ₂ , R ₃ and R ₄ are independently H or a hydrocarbon group. The hydrocarbon group is not limited, and examples thereof include an alkyl group or an alkyl group substituted with a hydroxyl group, a halogen atom, a nitro group, a carboxy group, an alkoxycarbonyl group, or a heteroaryl group. The number of carbon atoms in the hydrocarbon group is not limited, and examples thereof include C ₁ to C ₁₀ hydrocarbon groups.

  Moreover, this invention includes the prion protein structure conversion inhibitor which contains the said compound as an active ingredient. The prion protein structure conversion inhibitor is also an amyloid precursor special structure formation inhibitor that prevents the formation of infectious prion protein, and other conformation diseases such as Alzheimer's disease, Huntington's chorea, polyglutamine disease, type II diabetes, etc. It can also be applied to.

Furthermore, the present invention also encompasses a method of screening for in silico ligand investigated the specific binding of the PrP ^C structure prion potential candidate compound and the animal that bind to PrP ^C on the computer. Structure of PrP ^C prion example, F. Lopez Garcia, R. Zahn , R. Riek, K. Wuthrick, Proc. Natl. Acad. Sci. USA 97, 8334-8339 (2000) mouse, hamster, bovine, human The three-dimensional structure has been determined by NMR, and these structures can be used. The structure of mouse, hamster, cow and human are shown in R. Riek et al., Nature, 382, 180-182 (1996), TL James et al., Proc. Natl. Acad. Sci. USA 94, 10086-, respectively. 10091 (1997), F. Lopez Garcia, R. Zahn, R. Riek, K. Wuthrick, Proc. Natl. Acad. Sci. USA 97, 8334-8339 (2000) and L. Calzolai et al., Proc. Natl See Acad. Sci. USA 97, 8340-8345 (2000). By inputting all or part of the structure coordinates of PrP ^C into a computer running a computer program that expresses the three-dimensional structure coordinates of the molecule or a storage medium of the computer, a compound that can bind to PrP ^C is displayed on the computer. It becomes possible to screen with. Screening may be performed docking simulations of the candidate compound and PrP ^C. As software used for virtual ligand screening, commercially available software that can calculate docking may be used. Further, the docking simulation can be performed by a technique as disclosed in Japanese Patent Laid-Open Nos. 6-309385 and 7-133233. However, the present invention is not limited to these programs and methods.

Furthermore, the present invention encompasses optimizes the lead compound with the structure of GN8420, a method of screening for a compound capable of binding to the PrP ^C on the computer. The structure of GN8420 partially modified by introduction of substituents, selected that the coupling of the substituent introduced derivatives compound and PrP ^C was simulated with in silico by using the above software may bind do it.

A compound selected by in silico screening may be administered to cells infected with prions, and the expression level of PrP ^Sc upon administration may be measured. Compound binds to PrP ^C, the case of suppressing the structural conversion from PrP ^C to PrP ^Sc, the expression amount of PrP ^Sc is reduced.

Furthermore, the present invention includes a pharmaceutical composition comprising as an active ingredient a compound that suppresses the structural conversion of prion protein from normal prion (PrP ^C ) to infectious prion (PrP ^Sc ). The pharmaceutical composition is a prion protein structure conversion inhibitor containing, as an active ingredient, a compound that suppresses the structure conversion of the normal prion (PrP ^C ) of the present invention into an infectious prion (PrP ^Sc ). The inhibitor can be used for the prevention or treatment of diseases caused by prion protein abnormalities such as sheep scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, GSS, FFI, Kool, and mutant Jacob disease. . The pharmaceutical composition of the present invention may contain a commonly used adjuvant and various additives as necessary. Examples of such various additives include pharmaceutically acceptable organic solvents, stabilizers, excipients, solubilizing agents, emulsifying agents, buffering agents, soothing agents, preservatives, coloring agents, and the like. These may be added by a conventional method.

  The pharmaceutical composition of the present invention can be administered to animals such as humans, cattle, sheep, horses, etc. infected with prions by the usual administration route such as transdermal, oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, etc. It can be performed by an administration method such as injection. The dose and frequency of administration of the pharmaceutical composition of the present invention are not limited, but may be appropriately set according to the animal species to be administered, the age of the animal, symptoms, the administration route, and the like. For example, the dose may be selected as appropriate within a range of several μg to several tens of mg per kg of body weight, and administered several times a week to several times a day. In addition, the subject to which the composition of the present invention is administered is not limited to an animal already infected with an abnormal prion, and may be administered to an animal that is not infected. Can be prevented.

Furthermore, the present invention also includes foods and beverages containing a compound that suppresses the structural conversion of prion protein from normal prion (PrP ^C ) to infectious prion (PrP ^Sc ).

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

The materials and reagents used in this example were as follows.
Compounds selected by virtual ligand screening (VLS) are Aldrich Chemical Company, Inc. (Milwaukee, USA), G & J Research Chemicals, Ltd. (Devon, UK), Maybridge Chemical Company, Ltd. (Cornwall, UK), AsInEx Ltd. (Moscow, USSR). These compounds were dissolved in DMSO for in vivo screening and directly as a solution for spectral analysis for spectral analysis. Recombinant mouse prion protein (the 23rd to 231st amino acids) was obtained from PRIIONICS AG (Schlieren, Switzerland) and dissolved in H ₂ O.

This example was performed as follows.
[Virtual ligand screening]
ACD database for (MDLInformation Systems, Inc., San Leandro , CA) approximate compound of 320000 in mouse PrP ^C structure (F. Lopez Garcia, R. Zahn, R. Riek, K. Wuthrick, Proc. Natl. Acad. Sci. USA 97, 8334-8339 (2000)) was examined for specific binding with ligands in silico.

[Cells and antibodies]
GT1-7, an immortalized mouse neuronal cell line, was cultured in DMEM medium containing 4.5 g / ml glucose, 10% fetal calf serum (ICN, UK) and 1% streptomycin-penicillin (GIBCO / BRL) . Every 3 to 4 days, the cells were trypsinized, diluted 1: 5 and passaged. GT1-7 cells stably infected with Fukuoka-1 or 22L strain are described in O. Milhavet et al., Proc. Natl. Acad. Sci. USA 97, 13937-13942 (2000). Compound stock solutions (19 of 59 selected compounds) were prepared in 100 mM DMSO and stored at 4 ° C. Prior to use, the compounds were diluted with media. Control cells were treated with medium containing only solvent (0.1%). Approximately 2 × 10 ⁵ cells were placed in each well of a 6-well plate and drug treatment was started 15 hours after compound addition. After 72 hours of incubation, cells were lysed in 150 μl of 1 × Triton / DOC lysis buffer (0.5% tritonX-100, 0.5% deoxycholic acid, 150 mM NaCl, 25 mM Tris HCl pH7.5, 2 mM EDTA and pepstatin, leupepsin). It was. Protein concentration was measured by BCS assay (Pierce) and each sample was normalized to 2 mg protein. To analyze PrP ^sc production, proteins were digested with proteinase K for 30 minutes at 37 ° C. at a concentration of 10 μg / mg. Digestion was inhibited with PMSF (2 mM) and samples were subjected to SDS / PAGE using a 15% SDS / PAGE gel. The protein was then transferred to a PVDF membrane (Immobilon-P, Amasham, USA). Anti-mouse PrP antibody (SS28) was used for PrPres. For detection of PrP ^C , SAF32 antibody (SPI bio, France) was used as the primary antibody. The signal was visualized with ECL-plus (Amasham) and exposed to X-ray film (Konica). Signal quantification was performed by scanning the membrane with Fluorochem (Alpha Innotech, US).

[Modification by heat]
All circular dichroism measurements were made on an Aviv202 stopped flow circular dichroism spectrometer. The far ultraviolet spectrum was recorded between 80 and 250 nm using a cell with 1 nm slit width and 0.1 cm path length thermally stabilized at 25 ° C. Mouse PrP denaturation includes 5.0 μM PrP and samples with or without 10.0 μM candidate compounds measure the CD signal at 222 nm at a heating rate of 1 ° C./min over a temperature range of 25 to 85 ° C. Monitored by All measurements were performed in 100 mM sodium phosphate buffer pH 7.4. In order to confirm that the Tm value does not depend on the heating rate, the sample was heated at a heating rate of 2, 1, 0.5 ° C./min and thermally denatured. The heat denaturation profile was fitted with the following two-phase model (F. Lopez Garcia, R. Zahn, R. Riek, K. Wuthrick, Proc. Natl. Acad. Sci. USA 97, 8334-8339 (2000)).

ここで、RおよびTはそれぞれガス定数および温度（K）であり、Tmは融解温度であり、ΔHは熱変性に関連したエンタルピー変化であり、Yは222nmでのCDシグナルであり、YNおよびYUは、それぞれ天然および変性した形態のものによるシグナルであり、mNおよびmUhaは、それぞれ天然および変性した形態のもののスロープを示す。

Where R and T are the gas constant and temperature (K), respectively, Tm is the melting temperature, ΔH is the enthalpy change associated with heat denaturation, Y is the CD signal at 222 nm, YN and YU Are signals due to the natural and denatured forms, respectively, and mN and mUha indicate the slopes of the natural and denatured forms, respectively.

  In this case, a non-linear least squares routine of the Sigma Plot 2001 program (SPSS Inc. Chicago) was used.

From the above examination, the following results were obtained.
Of binding pocket of 5 points present in the PrP ^C, we particularly focused on green binding pocket located between the C-terminal region of the A-S2 loop and helix B of FIG. 1A. First, it searched from 320,000 compounds in the ACD database by computer docking simulation. The program optimizes the mobile ligand structure for the receptor site potential field of the mouse prion. 624 compounds having a binding energy of −32 kcal / mol or less were selected. Subsequently, the binding structure was further optimized by moving the side chain of the accepting site (M. Schapira, BM Raake, HH Samuels, and R. Abagyan, Proc. Natl. Acad. Sci. USA 97, 1008-1013). (2000)). After further careful examination, 59 candidates were selected. These were subjected to the following in vitro test. (6 of them are shown in the table)

Mouse hypothalamic neuronal lineage GT1 can infect mouse prions. In order to evaluate the anti-prion action of the compounds, we used the GT ^FK-1 cell line (N. Nishida et al., J. Virol, 74, 320-325 (2000)). These are Fukuoka-1 strains stably infected with mouse-compatible prions derived from GSS (O. Milhavet et al., Proc. Natl. Acad. Sci. USA, 97, 13937-13942 (2000)). Among the compounds tested, 72 hours with 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino) -benzyl] -phenyl] -acetamide, which we named GN8420 Treated cells showed a marked decrease in PrP ^Sc and only GN8420 was found to strongly suppress scrapie-type production in the GT ^FK-1 cell line (FIG. 2A, bottom panel). The other GN3 to GN7 had no effect as seen in FIG. Other candidates were highly cytotoxic. The effect of GN8420 was dose-dependent as seen in FIG. 2B, and the effective concentration (EC50) at which the amount of PrP ^Sc was halved was 1.35 μM when four experiments were repeated (FIG. 2B). . Furthermore, when treated with 5 μM for 7 days, PrP ^Sc disappeared completely. A similar anti-prion effect was also observed in GT22L cells persistently infected with scrapie-derived mouse prion 22L, which reduced PrP ^Sc at 10 μM (FIG. 2C). GN8420 reduced the scrapie type at 10 μM. This indicates that GN8240 is not specific for prion strains. In FIG. 2C, molecular weights (37, 25, 15 kDa) are shown on the right side of the panel. Interestingly, the expression level of PrP ^C in uninfected GT1-7 was relatively high even in cells treated with GN8420, and PrP ^C was stabilized to some extent at 10 μM (FIG. 2D). This is this medicine, as shown by computer simulation suggests that to actually stabilize the PrP ^C.

We further GN8420 is whether to directly stabilize the PrP ^C, was examined. FIG. 3A is a heat denaturation curve of a recombinant mouse prion protein (amino acids 23-231, 5 μM). Residue average molar ellipticity at 222 nm reflecting the helix content was plotted against temperature. Using the two-state model (GD Ramsay and MR Eftink, Methods Enzymol. 240, 615-645 (1994)), the transition temperature and enthalpy change were [65.3 ± 0.4 ° C and 35.0 ± in the absence of GN8420 (10 μM). 1.9 kcal / mol] and [67.7 ± 0.6 ° C and 41.8 ± 2.2 kcal / mol] in the presence. In FIG. 3A, white circles are the results in the presence of GN8420, and black circles are the results in the absence of GN8420. Interestingly, as seen in FIG. 3A, the increase in ellipticity around 40 ° C. seen in the absence of GN8420 was not observed in the presence of GN8420. Since an increase in ellipticity refers to an increase in helix content, this is thought to refer to intermediate formation prior to full denaturation. Therefore, GN8420 binds to PrP ^C, was found to suppress the intermediate formation. In contrast, other compounds containing GN4, such as GN4, did not inhibit intermediate formation.

In order to understand the mechanism of the intermediate inhibitory effect by GN8420 in more detail, we optimized the structure of the PrP ^C -GN8420 complex using a mobile side chain. As seen in Figure 3B, GN8420 specifically binds the pocket located between the A-S2 helices and loop B in mouse PrP ^C. As shown in FIG. 3B, GN8420 connects N159 (A-S2 loop) and E196 (BC loop) with five hydrogen bonds at distant residues. In contrast, N, N '-(methylenedi-4,1-phenylene) bis (3-methyl-1-piperidinecarboxamide, named GN4, did not inhibit PrP ^Sc production, but the neighboring residues (R156, N159, Q160) were found to be linked by hydrogen bonds only, as shown in Fig. 3C, showing long-range hydrogen bonds between GN8420 and mouse PrP ^C. Long-range hydrogen bonds are indicated by arrows in the figure. O1 (GN8420) -HN (N159) (2.05Å), O1 (GN8420) -Hδ22 (N159) (2.53Å), O1 (GN8420) -HN (Q160) (2.43Å), HO1 (GN8420) -Oε1 (E196) (2.43Å) and Fig. 3D shows the short-range hydrogen bond between GN4 and mouse PrP ^C. The short-range hydrogen bond is indicated by an arrow in the figure. That is, O2 (GN4) -Hε (R156) (2.59Å), O1 (GN4) -HN (N159) (2.13Å), O1 (GN4) -HN (Q160) (2.52Å) Other compounds that had no effect were also connected only by neighboring residues. Therefore, GN8420 cannot suppress the formation of PrP * by connecting the long-range remaining period with hydrogen bonds and the C-sheet helix. From these findings, GN8420 binds to PrP ^C , reduces the population of PrP ^* , and PrP ^Sc , as shown in FIG. In Fig. 4, PrP ^C , PrP ^* , PrP ^U and PrP ^Sc show a natural form (spherical form), an intermediate form, a denatured form, and a scrapie form, respectively, and GN8420 binds to PrP ^C. Then, the structural conversion of PrP ^C to PrP ^* inhibits the conformational fluctuations around the β-sheet and helix B and C. Since PrP ^* is an intermediate structure in the conversion of PrP ^C to PrP ^Sc , PrP ^* The decrease in the group had a decrease in PrP ^* Russ.

Mutations that cause some disease (SB Prusiner, Curr. Top. Microbiol. Immunol. 207, 1-17 (1996)) are confined to helices B and C. The helices B and C at the C end have low thermal stability under physiological conditions. Notably, it was observed that the vicinity of the interface between helix C and the beta sheet was extremely unstable. Furthermore, a C-terminal helix is necessary for infectivity (T. Muramoto, M. Scott, FE Cohen, SB Prusiner, Proc. Natl. Acad. Sci. USA 93, 15457-15462 (1996)). The N-terminal hydrophobic cluster part is known to cause neuronal apoptosis (G. Forloni et al., Nature 362, 543-546 (1993)). Therefore, it is considered that the interaction between the C-terminal helix and the N-terminal β sheet may be involved in the definitive structural transformation process. The hydrophobic cluster is in contact with S2 and helix B, and structural changes in this region may switch the structural transformation from PrP ^C to PrP ^Sc . We have found that the PrP molecule undergoes slow millisecond fluctuations, and that the fluctuation is along the path from PrP ^C to PrP ^* (K. Kuwata et al., Unpublished results). Therefore, whether GN8420 interferes with abnormal PrP accumulation by mutated genes would be a very interesting question.

Congo Red (B. Caughey and RE Race, J. Neurochem. 59, 768-771 (1992)), Amphotericin B (A. Mange et al., J. Virol. 74, 3135-3140 (2000)), Pentosansal Compounds such as fate (S. Supattapone et al., J. Virol. 75, 3453-3461 (2000)) are known to reduce or eliminate scrapie forms in cultured cells infected with prions. The molecular mechanism of the anti-prion action of these compounds is not well known. Recently, several independent groups have been developed in vitro (D. Peretz et al., Nature. 412, 739-43 (2001), M. Enari, E. Flechsig, C. Weissmann, Proc. Natl. Acad. Sci. USA 98, 9295-9299 (2001)) reported that anti-prion antibodies inhibit scrapie-type formation. The most studied 6H4 binds to the N-terminus of the helix and interferes with the interaction between PrP ^C and PrP ^Sc on the cell surface (M. Enari, E. Flechsig, C. Weissmann, Proc. Natl. Acad Sci. USA 98, 9295-9299 (2001)). On the other hand, GN8420 exerts an anti-prion action with a completely different action. That is, the weak structure is stabilized by bridging A-S2 and the BC loop. This demonstrates that the early changes in the molecule play an essential role in the pathological structural changes of PrP. By applying this discovery, it will be possible to design therapeutics for other conformational diseases such as prion disease and Alzheimer's disease caused by destabilization of the natural structure.

It is a figure which shows the structure of a mouse prion. It is a figure which shows the structure of GN8420. It is a figure which shows the structure of GN4. It is a figure which shows the effect with respect to the GT ^FK-1 cell of the different compounds (GN3, GN5, GN6, GN7, GN4, and GN8420) obtained from the computer screening. Cells treated with GN8420 for 72 hours show a marked decrease in PrP ^Sc . FIG. 2 shows the dose-dependent effect of GN8420 on GT ^FK-1 cells infected with GSS-derived mouse-matched prions. It is a figure which shows the dose dependence effect of GN8420 with respect to GT22L cell infected with scrapie origin mouse | mouth prion 22L. It is a figure which shows the effect of GN8420 with respect to GT1-7 which is not infected with prion. It is a figure which shows the heat denaturation curve of a mouse | mouth recombinant prion in presence or absence of GN8420 (10 micromol). It is a diagram illustrating a PrP ^C and GN8420 composite structure. It is a diagram showing a long-distance hydrogen bonds in PrP ^C and GN8420 composite structure. Long-range hydrogen bonds are indicated by arrows in the figure. It is a diagram showing a short-range hydrogen bonds in PrP ^C and GN8420 composite structure. The short-range hydrogen bond is the part indicated by the arrow in the figure. It is a figure which shows the mechanism in which GN8420 suppresses PrP ^Sc production | generation. When GN8420 binds to PrP ^C, helix B to beta sheet in PrP ^C, structure fluctuations around the C is strongly suppressed, intermediate, structural transformation to PrP ^* is suppressed. This results in a decrease in the population of PrP ^Sc .

Claims (10)

A compound that binds to the binding pocket of normal prion protein (PrP ^C ) and suppresses the production of folding intermediate prion protein (PrP ^* ), thereby suppressing the conversion of normal prion to infectious prion. The compound according to claim 1, wherein the binding pocket is present between the A-S2 loop of normal prion protein (PrP ^C ) and the C-terminal side of helix B. Formula (I)

(Where R ₁ , R ₂ , R ₃ and R ₄ are independently H or a hydrocarbon group)
The compound of Claim 1 or 2 represented by these. 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino)
The compound according to claim 3, which is -benzyl] -phenyl] -acetamide or a derivative thereof.   A prion protein structure conversion inhibitor comprising the compound according to any one of claims 1 to 4 as an active ingredient.   A preventive or therapeutic agent for prion diseases comprising the compound according to any one of claims 1 to 4 as an active ingredient.   The preventive or therapeutic agent for prion disease according to claim 6, wherein the prion disease is selected from the group consisting of sheep scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, GSS, FFI, Kuru and mutant Jacob disease.   A method for suppressing the conversion of a normal prion to an infectious prion, comprising preparing the compound according to any one of claims 1 to 4 and contacting the compound with a normal prion protein.   A computer having docking simulation software operable, a step of inputting a three-dimensional structure of a candidate compound into a computer storing a three-dimensional structure of a normal prion in a storage medium, and a normal prion on the computer A screening method for a compound capable of suppressing the structural transformation of a prion protein, comprising a step of performing docking simulation of a candidate compound in the previous period. Candidate compound is 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl
The screening method of the compound which can suppress the structural conversion of the prion protein of Claim 9 which is a derivative of -acetylamino) -benzyl] -phenyl] -acetamide.

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