JP2006030037A - Receptor ligand identifying method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To identify a compound for adjusting a receptor in a nucleus. <P>SOLUTION: The method of identifying the compound for adjusting the receptor in the nucleus comprises (A) a process of providing a candidate compound, and (B) a process of determining whether the candidate compound covalently bonds to cysteine in the receptor in the nucleus. The latter process includes a process of identifying the candidate compound that is determined to covalently bond as the compound for adjusting the receptor in the nucleus. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to methods and programs for obtaining agonists with higher affinity for or modulating nuclear receptors, and programs, and methods for producing such agonists. Furthermore, the present invention relates to a novel method for determining the affinity of a covalently bound agonist. Furthermore, the present invention relates to a novel method for determining the affinity of a non-covalent agonist using a covalently bound agonist.

  The present invention relates to a method and a program for obtaining an agonist having higher affinity for or modulating a nuclear receptor, and a program, and such an agonist, using the three-dimensional structure of a complex of a nuclear receptor and an agonist It relates to the manufacturing method. Furthermore, the present invention relates to a novel method for determining the affinity of a covalently bound agonist using the three-dimensional structure of a complex of a nuclear receptor and an agonist. Furthermore, the present invention relates to a novel method for determining the affinity of a non-covalent agonist using a covalently bonded agonist using the three-dimensional structure of a nuclear receptor / agonist complex.

  Furthermore, the present invention relates to a program for the above method.

  When cells respond to stimuli from outside the cell, various proteins within the cell are involved. Therefore, agonists and / or antagonists that target these proteins can be used as lead compounds for new drugs.

  Conventional methods for searching for agonists and / or antagonists of proteins include a search method using physical binding to a target protein as an index, and the ability to induce a biological reaction involving the target protein (function-inducing ability). There is a search method using as an index.

  The method using the physical binding to the target protein as an indicator examines how much the compound from the compound library (for example, combinatorial library) binds to the target protein and how many molecules bind to it. This is a method of selecting a compound having an affinity for a target protein that exceeds a certain evaluation standard. However, when screening from a compound library, it is necessary to screen a very large number of candidate compounds, which requires a great deal of labor and expense.

As a technique for screening an agonist having higher affinity from a compound library, a binding assay method using a [ ³ H] or [ ¹²⁵ I] labeled ligand is known (Non-patent Documents 1 and 2). However, even if such a method is used, there is no difference from the conventional method in terms of the types of compounds to be screened as candidate compounds. Therefore, the majority of compounds still need to be screened in the prior art to isolate more potent agonists. In addition, in a binding assay, a labeled compound is usually used, and for this purpose, a special facility or apparatus is required in order to use a radioisotope label as a ligand. In addition, there is a problem that a radioisotope-labeled ligand is expensive.

  Nuclear receptors were identified as those that act as transcription factors by forming a complex when bound by a ligand and binding to DNA. Nuclear receptors typically have six functional regions (domains) A to F (FIG. 1). The A / B region is thought to be involved in cell type-specific transcriptional activity, the C region is called a DNA-binding region having two Zinc finger structures, and the E / F region is called a ligand-binding domain. Involves in nuclear translocation and coactivator binding, and controls transcription. The first nuclear receptor found was a steroid hormone receptor to which steroid hormones bind, and the gene was cloned and sequenced in the mid 1980s, confirming that they were highly homologous to each other. Many homologous nuclear receptor-like proteins were subsequently discovered due to sequence homology. In human genome decoding, it is said that the number of similar proteins reaches 48 or more, and these are also called nuclear receptor superfamily.

  PPARγ, which is classified as a representative nuclear receptor, was identified in 1990 as a member of the nuclear receptor family represented by steroid hormone / thyroid hormone. PPARγ also has six functional regions A to F typical of the nuclear receptor family shown in FIG.

  Since the discovery that fibrates, which are antihyperlipidemic drugs, are agonists of PPARα, and thiazolidine derivatives known as antidiabetic agents are agonists of PPARγ, The search for PPARγ agonists following these compounds has been advanced, and attempts have been made to develop them as drugs for improving hyperlipidemia, insulin resistance, and diabetes (Non-patent Document 3). However, as described above, the screening of compound libraries requires a great amount of labor, time, and cost, and therefore, progress in the development of drugs for improving these PPARγ-related diseases is not sufficient.

  Other nuclear receptors are also thought to be factors that regulate the metabolism of lipids in the body, such as cholesterol, and various diseases associated with this metabolic disturbance, such as type II diabetes, obesity, hyperlipidemia, There is a great demand for facilitating ligand screening because it has attracted attention as related to many so-called lifestyle diseases (adult diseases) such as cardiovascular diseases.

Accordingly, it is desirable to develop methods for designing and / or making agonist derivatives with increased affinity from a given agonist.
Biochemistry, 41: 6640-, 2002 Nature, 402: 880-, 1999. WelVAS, Dec. 2001, no. 4, Cell Engineering No. 23, no. 2, 179-187

  It is an object of the present invention to provide a method for increasing the affinity of an agonist for a nuclear receptor when given an agonist for the nuclear receptor without substantially impairing the activity of the agonist. . It is also an object of the present invention to provide a method for designing an agonist with increased affinity using such a method. It is also an object of the present invention to provide agonists with increased affinity using such methods. It is also an object of the present invention to provide a program used in such a method, a storage medium including such a program, and a computer including such a program.

  It is also an object of the present invention to provide a method for designing a high affinity agonist for a nuclear receptor such as PPARγ according to the present invention.

  Furthermore, an object of the present invention is to provide a novel method for determining the affinity of a covalently binding agonist. Furthermore, it is an object of the present invention to provide a novel method for determining the affinity of a non-covalent agonist using a covalently bonded agonist.

  We have derivatized a given agonist so that it can be covalently bound to a nuclear receptor, increasing the affinity of the agonist for the nuclear receptor and increasing the affinity. In some cases, the present invention has been completed by finding that its action as an agonist can be enhanced.

  Accordingly, the present invention provides the following.

  In one aspect, the invention is a method of identifying a compound that modulates a nuclear receptor, the method comprising: A) providing a candidate compound; and B) the candidate compound comprising the nuclear receptor Providing a method comprising the step of determining whether to covalently bind to cysteine in the body, the method comprising identifying a candidate compound determined to be covalently bound as a compound that modulates a nuclear receptor To do.

  In one embodiment, the cysteine is present in the ligand binding domain.

  In another embodiment, the nuclear receptor is PPARα, PPARβ / δ, PPARγ1, PPARγ2, ERR1, ERR2, ERR3, HNF4α, HNF4β, RARα, RARβ, RARγ1, RARγ2, RXRα, RXRβ, RXRγ, RORα, RORβ , RORγ, LRH1, SF1, TLX and the O4245 receptor.

  In a preferred embodiment, the nuclear receptor is PPARγ.

  In one embodiment, the position of cysteine is in the following table:

It is shown by the amino acid number in the SEQ ID NO shown.

In another embodiment, the candidate compound is a compound having a substituent of R _A —CH═CH— (C═O) —R _B — or R _A — (C═O) —CH═CH—R _B —. Wherein R _A is a monovalent substituent containing hydrogen or an optionally substituted substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group, and R _B Is a divalent hydrocarbon group.

  In one embodiment, the covalent bond measurement is performed using fluorescently labeled maleimide.

  In another embodiment, the present invention further comprises determining whether the candidate compound hydrogen bonds with an amino acid in helix 12 or helix 5 in the ligand binding domain of the nuclear receptor.

  The residue number of the amino acid in helix 12 or helix 5 is

Arginine or tyrosine represented by the amino acid number in SEQ ID NO.

  In another embodiment, the amino acid in helix 12 or helix 5 comprises tyrosine 473 of the amino acid sequence shown in SEQ ID NO: 42 or tyrosine 501 of the amino acid sequence shown in SEQ ID NO: 44.

  In another embodiment, the hydrogen bond with the specific amino acid is measured by means of a nuclear magnetic resonance apparatus (NMR) or an X-ray diffractometer.

  In another embodiment, the covalent bond with the specific amino acid is measured by means of a mass spectrometer or SDS-PAGE.

In another aspect, the invention is a compound that can be identified by the method of the invention, wherein the compound has the following formula:
(Chemical formula 4)
R ₁ —CH═CH— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —CH═CH—R ₂ —R ₃
R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 13 carbon atoms connecting the shortest distance, and R ₂ is substituted A saturated or unsaturated hydrocarbon group, and R ₃ is monovalent containing hydrogen or a substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group, which may be substituted. Here, R ₂ and R ₃ provide a compound in which the maximum number of carbon atoms connecting the shortest distance is 4 to 14 for the contained carbon.

In one embodiment, in the above compound, R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 9 carbon atoms connecting the shortest distance, and R ₂ Is an optionally substituted saturated or unsaturated hydrocarbon group, and R ₃ is a monovalent hydrocarbon group containing a substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group and a thiol group Where R ₂ and R ₃ are 6 to 12 carbon atoms having the shortest distance between the contained carbon atoms.

In another aspect, the present invention is a composition for modulation of a nuclear receptor comprising a compound identified by the method of the present invention, said compound having the formula:
(Chemical formula 5)
R ₁ —CH═CH— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —CH═CH—R ₂ —R ₃
R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 13 carbon atoms connecting the shortest distance, and R ₂ is substituted A saturated or unsaturated hydrocarbon group, and R ₃ may be hydrogen or a monovalent containing a substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group. Here, R ₂ and R ₃ provide a composition in which the maximum value of the number of carbons connecting the shortest distance is 4 to 14 for the contained carbon.

In another embodiment, the structure is
R ₁ — (C═O) —CH═CH—R ₂ —R ₃ ,
In the above compound,
R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 9 carbon atoms connecting the shortest distance, and R ₂ is an optionally substituted saturated or unsaturated hydrocarbon group. A saturated hydrocarbon group, R ₃ is a monovalent substituent containing hydrogen or an optionally substituted substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group; Here, for R ₂ and R ₃ , the maximum number of carbons connecting the shortest distance is 6 to 14 for the contained carbon, and the combination is as follows:

To the nuclear receptor having the amino acid sequence shown in SEQ ID NO.

In another embodiment, the present invention is a composition for the treatment, prevention or prognosis of a disease, disorder or condition caused by a nuclear receptor comprising a compound identified by the method of the present invention, comprising: The above compound has the following formula:
(Chemical formula 6)
R ₁ —C═C— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —C═C—R ₂ —R ₃
Where R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 13 carbon atoms connecting the shortest distance, and R ₂ is A saturated or unsaturated hydrocarbon group which may be substituted, and R ₃ represents a substituent selected from the group consisting of hydrogen or an optionally substituted carbonyl group, hydroxyl group, amide group and thiol group; In the present invention, R ₂ and R ₃ provide a composition having a maximum number of carbon atoms connecting the shortest distance of 4 to 14 carbon atoms.

  In one aspect, the present invention provides A) a step of mixing a target biomolecule and a ligand to form a mixture; B) a step of adding a fluorescent-labeled maleimide to the mixture; C) the target biomolecule in the mixture. There is provided a method for quantifying the binding ability of a ligand to a target biomolecule, which comprises the step of detecting the binding between the fluorescent label maleimide and the fluorescent label maleimide.

  In one embodiment, the present invention further comprises a step of performing the quantification of the covalent bond, wherein the binding is a covalent bond, and the quantification is achieved by measuring at least two amounts with respect to the ligand. The

  In another embodiment, ligands include those that are covalently bonded or those that are not covalently bonded.

In other embodiments, the biomolecule used in the present invention is a nuclear receptor. Here, preferably, the ligand to irreversibly covalent bonds, indomethacin, 15-deoxy - [delta ^{12, 14} - prostaglandin _J 2 (11- oxo - prostaglandin -5Z, 9,12E, 14Z- tetraene-1 Oic acid; 15d-PGJ ₂ ), 11-oxo-prosta-5Z, 12E, 14Z-triene-1-oic acid (CAY10410), 5-oxo-6E, 8Z, 11Z, 14Z-eicosatetraenoic acid (5 -OxoODE), 9-oxo-10E, 12Z-octadecanodienoic acid (9-oxoODE), 13-oxo-9Z, 11E-octadecanodienoic acid (13-oxoODE) and 15-oxo-5Z , 8Z, 11Z, 13E-Eiocosatetraenoic acid (5-oxoETE) selected from the group consisting of Indomethacin has the following structural formula.

  In another embodiment, the invention is a system for identifying a compound that modulates a nuclear receptor, the system comprising: A) whether a candidate compound is covalently bound to a cysteine in the nuclear receptor. And B) means for calculating a candidate compound determined to be covalently bound as a compound that modulates a nuclear receptor.

  In another aspect, the present invention relates to a kit for measuring the covalent bond between a compound and the nuclear receptor, which is used for identifying a compound that modulates a nuclear receptor, comprising A) the above A kit is provided comprising: a ligand known to irreversibly covalently bind to a nuclear receptor; and B) instructions describing a procedure for measuring binding of a ligand that is not covalently bound.

  In yet another aspect, the present invention is a program for causing a computer to execute a method for identifying a compound that modulates a nuclear receptor, the method comprising: A) providing three-dimensional structure data of a candidate compound; and B) determining whether the candidate compound is covalently bound to cysteine in the nuclear receptor, wherein the candidate compound determined to be covalently bound is a compound that modulates a nuclear receptor A program including an identifying step is provided.

  In still another aspect, the present invention provides a recording medium storing a program for causing a computer to execute a method for identifying a compound that modulates a nuclear receptor, the method comprising: A) Three-dimensional structure data of a candidate compound And B) determining whether the candidate compound is covalently bound to cysteine in the nuclear receptor, wherein the candidate compound determined to be covalently bound modulates the nuclear receptor A recording medium comprising the step of identifying the compound as

  In addition, in accordance with the present invention, a method is provided that, when given an agonist for a nuclear receptor, increases the affinity of the agonist for the nuclear receptor without substantially compromising the activity of the agonist. Also provided are methods of designing agonists with increased affinity using such methods. Further, using such methods, agonists with increased affinity are provided. A program used in such a method, a storage medium containing such a program, and a computer containing such a program are also provided.

  In certain embodiments, methods of designing high affinity agonists for PPARγ are provided according to the present invention. The agonist designed in the present invention is expected to have a therapeutic effect by activating nuclear receptors, and is expected to have a therapeutic effect on many metabolic syndromes. For example, there is a report that the development of arteriosclerotic lesions was suppressed by administration of the PPARγ agonist triglitazone to genetically hyperlipidemic rabbits, LDL receptors under high fat diets, and apoE knockout mice. The compound of the present invention having high operation efficiency is expected to exert a higher therapeutic effect in arteriosclerosis.

In certain embodiments, triglitazone, 15d-PGJ ₂ inhibits secretion of macrophage phagocytosis, scavenger receptor class A involved in bactericidal action, inducible NO synthase, TNF-α, IL-1α, IL-6 Therefore, the compound of the present invention having high operation efficiency is expected to exhibit an anti-inflammatory action (= control of macrophage function) and exhibit a higher therapeutic effect.

  In certain embodiments, since PPARγ plays an important role in adipocyte differentiation, and PPARγ agonists improve insulin-resistant diabetes, the compounds of the present invention with high agonist efficiency may be used for fat metabolism and sugar metabolism. It is expected to exert a high therapeutic effect on obesity and diabetes caused by abnormalities.

  In a specific embodiment, since it is not necessary to label the ligand with a radioisotope label or a fluorescent label, screening of a new drug and quantification of the binding ability of the ligand can be performed easily and inexpensively. For example, in accordance with the present invention, a novel method for determining the affinity of a covalently bound agonist is also provided. Furthermore, in accordance with the present invention, there is provided a novel method for determining the affinity of a non-covalent agonist using a covalently bound agonist.

  In another aspect, the present invention provides a method for determining whether a candidate compound is an agonist or antagonist of a nuclear receptor. The method includes: A) providing a candidate compound; and B) determining whether the candidate compound is covalently bound to a cysteine in the nuclear receptor and selecting the candidate compound determined to be covalently bound C) determining the activity of the candidate compound in a system comprising a nucleic acid construct comprising a nucleic acid sequence encoding a reporter operably linked to a transcription factor recognition sequence and the selected nuclear receptor. When the expression of the reporter is enhanced, the candidate compound is determined to be an agonist of the nuclear receptor, and when the expression of the reporter decreases, the candidate compound is an antagonist of the nuclear receptor The step of determining.

  In one embodiment, the reporter used in the present invention is preferably luciferase.

  In another embodiment, the system used in the present invention is a cell.

  Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  According to the present invention, a ligand of a nuclear receptor can be identified with a high probability by checking the presence or absence of a specific simple binding. By using this screening method, high-throughput nuclear receptor ligand screening becomes possible. The present invention also provides specific nuclear receptor ligands. Such ligands have been found to be nuclear receptor ligands and thus proved useful in the treatment of various lifestyle diseases.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  The term “nuclear receptor” as used herein refers to a molecule that initiates a biological reaction in the nucleus by binding to a ligand. Usually, the nuclear receptor is present in the nucleus, but includes those having the property of moving into the nucleus when a biological reaction is initiated. Nuclear receptors usually form complexes when bound to bind to DNA and act as transcription factors.

  Here, biological reactions of nuclear receptors typically include transcriptional regulation (for example, promotion, suppression, initiation, or termination of transcription, which is typically promotion of transcription, but is not limited thereto. Is not). Typically, nuclear receptors are naturally present in the cytoplasm when unstimulated by a ligand and, when bound to the ligand, translocate into the nucleus and transmit the stimulus into the nucleus. Refers to molecules that Alternatively, a nuclear receptor initiates a biological reaction in the nucleus by binding to a ligand without changing its localization. Nuclear receptors include PPARα, PPARβ / δ, PPARγ1, PPARγ2, ERR1, ERR2, ERR3, HNF4α, HNF4β, RARα, RARβ, RARγ1, RARγ2, RXRα, RXRβ, RXRγ, RORα, RORβ, ROR1, RORβ, ROR1 , TLX and 04245 receptors, vitamin D receptors, thyroid hormone receptors, other steroid hormone receptors, and the like.

  These have a correspondence as described in the following table.

The above table shows the correspondence between abbreviations and international uniform names. O4245 has not yet been given a unified name. PPARγ1, PPARγ2, and RARγ1, RARγ2 have the same unified name because they are made by splicing differences from the same gene.

  As used herein, the term “PPARα” is an abbreviation for “peroxisome proliferator-activator receptor α” and means one type of nuclear receptor. PPARα is defined by the sequences shown in SEQ ID NOs: 37 and 38 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “PPARβ / δ” is an abbreviation for “peroxisome proliferator-activated receptor β / δ” and means one of the nuclear receptors. PPARβ / δ is defined by the sequences shown in SEQ ID NOs: 39 and 40 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “PPARγ1” is an abbreviation for “peroxisome proliferator-activated receptor γ1” and means one of the nuclear receptors. PPARγ1 is defined by the sequences shown in SEQ ID NOs: 41 and 42 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “ERR1” is an abbreviation for “Estrogen-related receptor 1” and means one of the nuclear receptors. ERR1 is defined by the sequences shown in SEQ ID NOs: 1 and 2 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “ERR2” is an abbreviation for “Estrogen-related receptor 2” and refers to one of the nuclear receptors. ERR2 is defined by the sequences shown in SEQ ID NOs: 3 and 4 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “ERR3” is an abbreviation for “Estrogen-related receptor 3” and means one of the nuclear receptors. ERR3 is defined by the sequences shown in SEQ ID NOs: 5 and 6 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “SF1” is an abbreviation for “Steroidogenic factor-1” and means one of nuclear receptors. SF1 is defined by the sequences shown in SEQ ID NOs: 9 and 10 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RXRα” is an abbreviation for “retinoid X receptor α” and means one of the nuclear receptors. RXRα is defined by the sequences shown in SEQ ID NOs: 11 and 12 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RXRβ” is an abbreviation for “retinoid X receptor β” and refers to one of the nuclear receptors. RXRβ is defined by the sequences shown in SEQ ID NOs: 13 and 14 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RXRγ” is an abbreviation for “retinoid X receptor γ” and means one type of nuclear receptor. RXRγ is defined by the sequences shown in SEQ ID NOs: 15 and 16 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “HNF4α” is an abbreviation for “hepatocyte nuclear factor 4α” and refers to one of the nuclear receptors. HNF4α is defined by the sequences shown in SEQ ID NOs: 17 and 18 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “HNF4γ” is an abbreviation for “hepatocyte nuclear factor 4γ” and refers to one of the nuclear receptors. HNF4γ is defined by the sequences shown in SEQ ID NOs: 19 and 20 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RARα” is an abbreviation for “retinoic acid receptor α” and refers to one of the nuclear receptors. RARα is defined by the sequences shown in SEQ ID NOs: 21 and 22 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RARβ” is an abbreviation for “retinoic acid receptor β” and refers to one of the nuclear receptors. RARβ is defined by the sequences shown in SEQ ID NOs: 23 and 24 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RARγ1” is an abbreviation for “retinoic acid receptor γ1” and means one type of nuclear receptor. RARγ1 is defined by the sequences shown in SEQ ID NOs: 25 and 26 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RARγ2” is an abbreviation for “retinoic acid receptor γ2” and means one of the nuclear receptors. RARγ2 is defined by the sequences shown in SEQ ID NOs: 27 and 28 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RORα” is an abbreviation for “RAR-related orphan receptor α” and means one of the nuclear receptors. RXRα is defined by the sequences shown in SEQ ID NOs: 29 and 30 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RORβ” is an abbreviation for “RAR-related orphan receptor β” and means one of the nuclear receptors. RXRβ is defined by the sequences shown in SEQ ID NOs: 31 and 32 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “RORγ” is an abbreviation for “RAR-related orphan receptor γ” and means one type of nuclear receptor. RXRγ is defined by the sequences shown in SEQ ID NOs: 33 and 34 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “O4245” is an abbreviation for “orphan receptor 4245” and refers to one of the nuclear receptors. O4245 is defined by the sequences shown in SEQ ID NOs: 35 and 36 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  As used herein, the term “TLX” is an abbreviation for “Talesless homolog” and refers to one of the nuclear receptors. TLX is defined by the sequences shown in SEQ ID NOs: 51 and 52 (nucleic acid sequence and amino acid sequence, respectively). In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity.

  Such receptors are described, for example, in Robinson-Rechavi M et al., J Cell Sci. It is understood that variants may be included so long as they have at least one activity as described in 2003 Feb 15; 116 (Pt 4): 585-586 and references cited therein.

  As used herein, a “biological activity” of a nuclear receptor (eg, PPARγ) is the activity provided by the nuclear receptor when the nuclear receptor binds to its ligand; Examples thereof include, but are not limited to, activities as described immediately before in the present specification (for example, PPARγ, promotion of transcription of PEPCK, promotion of gluconeogenesis, etc.). An agonist of a nuclear receptor (eg, PPARγ) induces at least a part or all of the biological activity of the nuclear receptor by binding to the nuclear receptor.

  As used herein, an “active” nuclear receptor (eg, PPARγ) refers to a form of the nuclear receptor that exerts biological activity that is not exerted in the absence of an agonist upon binding of an agonist. . Thus, for example, an agonist-bound nuclear receptor (eg, PPARγ) is an active nuclear receptor (eg, active PPARγ).

  The term “ligand” as used herein is a binding partner for a specific receptor or family of receptors. The ligand can be an endogenous ligand for the receptor, or alternatively, a synthetic ligand for the receptor, such as a drug, drug candidate, or pharmacological means.

  As used herein, the term “ligand binding site” refers to a moiety to which a ligand binds in a nuclear receptor. An “agonist binding site” is a doctrine “ligand binding site”. A ligand binding site includes amino acid residues in a polypeptide that have some interaction with a ligand, including but not limited to covalent bonds, hydrogen bonds, hydrophobic interactions, electrostatic interactions, and the like.

  The agonist binding site of the nuclear receptor can include a cysteine residue facing the agonist. PPARγ contains cysteine residues as listed in Table 4 as cysteine residues facing the agonist. For example, the alignment of the nuclear receptors other than PPARγ and the primary structure of PPARγ is performed as shown in FIG.

  As used herein, an “agonist” is a type of ligand that, when bound to a receptor, exhibits the same or similar effect as when the natural ligand of that receptor binds to the receptor. It refers to the factor.

As agonists for PPARγ, for example, non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, salicylic acid, ibuprofen, fenprofen, flufenamic acid, piroxicam, acetaminophen, 15-deoxy-Δ ^12,14 -prostaglandin J ₂ (11-oxo-prosta-5Z, 9,12E, 14Z-tetraene-1-oic acid; 15d-PGJ ₂ ), 11-oxo-prosta-5Z, 12E, 14Z-triene-1-oic acid (CAY10410) ), MCC-555, 9 (S) -hydroxyoctadecadienoic acid (9 (S) -HODE), 9-oxo-10E, 12Z-octadecanodienoic acid (9-oxoODE), o-lysophosphadi Tinic acid (o-LPA), 13 (S) -hydroxyoctade Cadienoic acid (13 (S) -HODE), 13-oxo-9Z, 11E-octadecanodienoic acid (13-oxoODE), eicosapentaenoic acid (EPA), dihomo-γ-linolenic acid (DGLA), arachidonic acid However, it is not limited to these.

  In the present specification, the “antagonist” is a kind of ligand, which acts antagonistically on the binding of a biologically active substance (ligand) to a receptor, and itself exhibits a physiological action via the receptor. Refers to no factors. Antagonists, blockers (blockers), inhibitors (inhibitors) and the like are also encompassed by this antagonist. Examples of antagonists for PPARγ include, but are not limited to, T0070907.

  As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also be assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is included.

  In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art. The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to in this specification are in L form. The term “unnatural amino acid” refers to an amino acid that is not normally found in proteins in nature. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine. “Amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally accepted single letter code.

The character codes are as follows.
3 letter code of amino acid 1 letter code Meaning Ala A alanine Cys C cysteine Asp D aspartic acid Glu E glutamic acid Phe F phenylalanine Gly G glycine His H histidine Ile I isoleucine Lys K lysine Leu L leucine Met M methionine Asl G Glutamine Arg R Arginine Ser S Serine Thr T Threonine Val V Valine Trp W Tryptophan Tyr Y Tyrosine Asx Asparagine or Aspartate Glx Glutamine or Glutamate Xaa Unknown or other amino acids.
Base symbol Meaning
a adenine g guanine c cytosine t thymine u uracil r guanine or adenine purine y thymine / uracil or cytosine pyrimidine m adenine or cytosine amino group k guanine or thymine / uracil keto group s guanine or cytosine w adenine or thymine / uracil b guanine or cytosine Thymine / uracil d adenine or guanine or thymine / uracil h adenine or cytosine or thymine / uracil v adenine or guanine or cytosine n adenine or guanine or cytosine or thymine / uracil, unknown or other base.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool.

  In the present specification, a “corresponding” amino acid has or is predicted to have the same action as a predetermined amino acid in a protein or polypeptide as a reference for comparison in a protein molecule or polypeptide molecule. For example, in the nuclear receptor, the domain corresponding to the A domain is a region having a transcription promoting function, and the domain corresponding to the B domain is a region having a transcription promoting function, Sometimes referred to as A / B domain. The domain corresponding to the C domain is a region having an action of DNA binding, and usually there are two characteristic Zn finger structures found in DNA binding proteins. The domain corresponding to the D domain is a region existing between the C domain and the E · F domain, and the domain corresponding to the E · F domain is a region having an action of binding to a ligand (hormone). Also called binding domain. This ligand binding domain (E / F domain) has a similar steric structure consisting of 12 α helices (FIG. 1). The domain corresponding to the F domain is a region existing on the C-terminal side from the E domain. In the case of an enzyme molecule, it refers to an amino acid that exists at the same position in the active site and contributes similarly to the catalytic activity. Accordingly, as used herein, in the amino acid sequence of a given nuclear receptor, the “amino acid residue corresponding to the tyrosine residue at position 473 of PPARγ” refers to the amino acid sequence of the given nuclear receptor When the amino acid sequences of PPARγ are aligned, these amino acid residues are aligned with the tyrosine residue at position 473 of PPARγ, and the predetermined nuclear receptor exhibits biological activity by hydrogen bonding with an agonist. Residues that are predicted to exert. Such corresponding residues can be identified by performing an alignment. In the present specification, A to F domains in nuclear receptors other than those listed above may correspond to domains in the range of amino acid positions corresponding to the first amino acid and the last amino acid in the above specific examples. Understood.

  Herein, the A to F domains can be determined based on definitions known in the art. For example, such a definition is described in Robinson-Rechavi M et al., J Cell Sci. 2003 Feb 15; 116 (Pt 4): 585-586 and the literature cited therein can be referred to.

  A comparison of representative domain alignments is shown in FIG. 2 (FIGS. 2-1 to 2-3).

  Therefore, as used herein, in the amino acid sequence of a given nuclear receptor, for example, the “corresponding amino acid residue” to the tyrosine residue at position 473 of PPARγ is the term “the corresponding amino acid residue”. When the amino acid sequence is aligned with the amino acid sequence of PPARγ, the amino acid residue is aligned with the tyrosine residue at position 473 of PPARγ, and the predetermined nuclear receptor is biologically bonded by hydrogen bonding with an agonist. Residues that are predicted to exert specific activity. Examples of such residues include:

However, it is not limited to these. As used herein, the “corresponding amino acid residue” for the cysteine residue at position 285 of PPARγ in the amino acid sequence of a given nuclear receptor refers to the amino acid sequence of that given nuclear receptor and the PPARγ amino acid sequence. When the amino acid sequences are aligned, the amino acid residues are aligned at the same position as the cysteine residue at position 285 of PPARγ, for example,

Etc., but are not limited thereto.

  These correspondences can also be determined by referring to FIGS. 2-1 to 2-3.

  As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. A “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2′-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide. Is an N3′-P5 ′ phosphoramidate bond derivative oligonucleotide, a ribose and phosphodiester bond in the oligonucleotide converted to a peptide nucleic acid bond, uracil in the oligonucleotide is C− A derivative oligonucleotide substituted with 5-propynyluracil, a derivative oligonucleotide where uracil in the oligonucleotide is substituted with C-5 thiazole uracil, and cytosine in the oligonucleotide is C-5 propynylcytosine Substituted derivative oligonucleotides, derivative oligonucleotides in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, derivative oligonucleotides in which the ribose in the DNA is replaced with 2'-O-propylribose Examples thereof include derivative oligonucleotides in which ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)). The term “nucleic acid” is also used herein interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. Particular nucleic acid sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.

  In the present specification, the “nucleotide” may be natural or non-natural. “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). .

  In the present specification, the “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. As used herein, preferably a receptor “fragment” specifically binds a ligand to which a full-length receptor can specifically bind.

  The protein used in the present invention may be purified from a natural source or may be recombinantly expressed by genetically engineering a host cell. Examples of a method for producing a polypeptide by genetic engineering include, for example, culturing a prokaryotic bacterium that produces the polypeptide, accumulating the recombinant receptor protein as inclusion bodies in the bacterium, and destroying the host bacterium. To obtain the polypeptide.

  “Transformant” refers to all or part of an organism such as a cell produced by transforming a host cell. A prokaryotic cell is illustrated as a transformant. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject, and encompasses all of these forms herein, but may refer to a particular form in a particular context. .

  The host bacterial cell for obtaining the transformant is not particularly limited as long as it expresses a polypeptide having physiological activity, and various host bacterial cells conventionally used in genetic manipulation can be used. . Examples of prokaryotic cells include prokaryotic cells belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacteria, Pseudomonas, etc., such as Escherichia coli XL1-Blue, Escherichia coli XL2-Blue. , Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 (DE3), Escherichia coli BL21 (DE3) pLysS, Escherichia coli HMS174 (DE3), Escherichia coli HMS174 (DE3) pLysS, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium ammoniagenes, Brevibacterium immm ariophilum ATCC14068, Brevibacterium saccharolyticumATCC14066, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp. Examples thereof include D-0110.

  As long as the polypeptide derived from the cell obtained in the present invention has substantially the same action as the naturally occurring polypeptide, one or more amino acids in the amino acid sequence are substituted, added and / or deleted. Alternatively, the sugar chain may be substituted, added and / or deleted.

  Certain amino acids can be substituted for other amino acids in a protein structure, such as, for example, the binding site of a ligand molecule, without an apparent reduction or loss of interaction binding capacity. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, a protein equivalent in ligand binding capacity). It is well known. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present invention, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine; However, it is not limited to these.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Since orthologs are useful for the estimation of molecular phylogenetic trees, orthologs may also be useful in the present invention.

  “Conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. The addition of amino acids means that one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids are added to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method or a hybridization method.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such a number is a function (for example, cancer marker, neurological disease) in a variant having the substitution, addition or deletion. As long as the marker etc.) is retained. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

(organic chemistry)
In the present invention, the hydrocarbon used in the compound is used in the broadest sense in the art, and may include unsaturated and saturated hydrocarbons, and variants thereof. Examples of such hydrocarbons include, for example, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkylene, optionally substituted cycloalkyl, and optionally substituted. Examples thereof include, but are not limited to, good alkenylene, optionally substituted cycloalkenyl, optionally substituted alkynyl, and optionally substituted carbocyclic group. It is understood that the substituent can include any of the substituents described herein.

In this specification, “a monovalent substituent containing a substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group, which may be substituted” refers to a carbonyl group, a hydroxyl group, and an amide group. Alternatively, it refers to a substituent having at least one functional group of a thiol group as a substituent, and a part of which may further have a substituent. Examples of such substituents include, but are not limited to, divalent hydrocarbons such as alkylene. Examples of such include, for example, HC (═O) —R _X (wherein R _X can be a single bond or a divalent substituent such as an alkylene group), H—O—R. _Y (where R _Y can be a single bond or a divalent substituent such as an alkylene group), R _Z1 —N (—R _Z2 ) —R _Z3 (where Z ₁ and Z ₂ are It can be a monovalent substituent such as hydrogen or an alkyl group, and R _Z3 can be a single bond or a divalent substituent such as an alkylene group), H—S—R _W (where R _W is And can be a single bond or a divalent substituent such as an alkylene group). For example, it is understood that the carbonyl group which may be substituted includes a carboxyl group.

As used herein, “alkyl” refers to a monovalent group formed by loss of one hydrogen atom from an aliphatic hydrocarbon (alkane) such as methane, ethane, or propane, and is generally represented by C _n H _{2n + 1} −. Where n is a positive integer. Alkyl can be linear or branched. In the present specification, the “substituted alkyl” refers to an alkyl in which H of the alkyl is substituted with a substituent specified below. Specific examples thereof include C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl. C1-C11 alkyl or C1-C12 alkyl, C1-C2 substituted alkyl, C1-C3 substituted alkyl, C1-C4 substituted alkyl, C1-C5 substituted alkyl, C1-C6 substituted alkyl C1-C7 substituted alkyl, C1-C8 substituted alkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11 substituted alkyl or C1-C12 substituted alkyl obtain. Here, for example, C1-C10 alkyl means linear or branched alkyl having 1 to 10 carbon atoms, methyl (CH _3- ), ethyl (C ₂ H _5- ), n-propyl. _{(CH 3 CH 2 CH 2 -} ), isopropyl _{((CH 3) 2 CH -} ), n- butyl _{(CH 3 CH 2 CH 2 CH} 2 -), n- pentyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 -), n-hexyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 -), n- heptyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 -), n- octyl _(CH 3 CH _{2 CH 2 CH 2 CH 2 CH} 2 CH 2 CH 2 -), n- nonyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 CH 2 CH 2 -), n- decyl _(CH 3 CH 2 CH ₂ C _{H 2 CH 2 CH 2 CH 2} CH 2 CH 2 CH 2 -), - C (CH 3) 2 CH 2 CH 2 CH (CH 3) 2, -CH 2 CH (CH 3) such as ₂ are exemplified. In addition, for example, C1-C10 substituted alkyl refers to C1-C10 alkyl, in which one or more hydrogen atoms are substituted with a substituent.

  As used herein, “optionally substituted alkyl” means that either “alkyl” or “substituted alkyl” as defined above may be used.

As used herein, “alkylene” refers to a divalent group formed by losing two hydrogen atoms from an aliphatic hydrocarbon (alkane) such as methylene, ethylene, and propylene, and is generally represented by —C _n H _2n —. Where n is a positive integer. The alkylene can be straight or branched. In the present specification, the “substituted alkylene” refers to an alkylene in which H of the alkylene is substituted with a substituent specified below. Specific examples thereof include C1-C2 alkylene, C1-C3 alkylene, C1-C4 alkylene, C1-C5 alkylene, C1-C6 alkylene, C1-C7 alkylene, C1-C8 alkylene, C1-C9 alkylene, C1-C10 alkylene. C1-C11 alkylene or C1-C12 alkylene, C1-C2 substituted alkylene, C1-C3 substituted alkylene, C1-C4 substituted alkylene, C1-C5 substituted alkylene, C1-C6 substituted alkylene C1-C7 substituted alkylene, C1-C8 substituted alkylene, C1-C9 substituted alkylene, C1-C10 substituted alkylene, C1-C11 substituted alkylene or C1-C12 substituted alkylene obtain. Here, for example, C1-C10 alkylene means linear or branched alkylene having 1 to 10 carbon atoms, methylene (—CH ₂ —), ethylene (—C ₂ H ₄ —), n - propylene _{(-CH 2 CH 2 CH 2 -} ), isopropylene _{(- (CH 3) 2 C} -), n- butylene _{(-CH 2 CH 2 CH 2 CH} 2 -), n- pentylene _(-CH 2 CH _{2 CH 2 CH 2 CH 2 -} ), n- hexylene _{(-CH 2 CH 2 CH 2 CH} 2 CH 2 CH 2 -), n- heptylene _{(-CH 2 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 - ), N-octylene (—CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ —), n-nonylene (—CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -), n-decylene _{(-CH 2 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -), - CH 2 C (CH 3) 2 - and the like are exemplified. Further, for example, C1-C10 substituted alkylene refers to C1-C10 alkylene, in which one or more hydrogen atoms are substituted with a substituent. As used herein, “alkylene” may contain one or more atoms selected from an oxygen atom and a sulfur atom.

  As used herein, “optionally substituted alkylene” means that either “alkylene” or “substituted alkylene” as defined above may be used.

  As used herein, “cycloalkyl” refers to alkyl having a cyclic structure. “Substituted cycloalkyl” refers to a cycloalkyl in which H of the cycloalkyl is substituted by the substituent specified below. Specific examples include C3-C4 cycloalkyl, C3-C5 cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl, C3-C10 cycloalkyl, C3-C11. Cycloalkyl, C3-C12 cycloalkyl, C3-C4 substituted cycloalkyl, C3-C5 substituted cycloalkyl, C3-C6 substituted cycloalkyl, C3-C7 substituted cycloalkyl, C3-C8 substituted Cycloalkyl, C3-C9 substituted cycloalkyl, C3-C10 substituted cycloalkyl, C3-C11 substituted cycloalkyl or C3-C12 substituted cycloalkyl. For example, cycloalkyl is exemplified by cyclopropyl, cyclohexyl and the like.

  In the present specification, the “optionally substituted cycloalkyl” means either “cycloalkyl” or “substituted cycloalkyl” as defined above.

As used herein, “alkenyl” refers to a monovalent group formed by losing one hydrogen atom from an aliphatic hydrocarbon having one double bond in the molecule, and is generally represented by C _n H _2n−1 —. (Where n is a positive integer greater than or equal to 2). “Substituted alkenyl” refers to alkenyl in which H of alkenyl is substituted by a substituent specified below. Specific examples include C2-C3 alkenyl, C2-C4 alkenyl, C2-C5 alkenyl, C2-C6 alkenyl, C2-C7 alkenyl, C2-C8 alkenyl, C2-C9 alkenyl, C2-C10 alkenyl, C2-C11 alkenyl or C2-C12 alkenyl, C2-C3 substituted alkenyl, C2-C4 substituted alkenyl, C2-C5 substituted alkenyl, C2-C6 substituted alkenyl, C2-C7 substituted alkenyl, C2-C8 substituted Alkenyl, C2-C9 substituted alkenyl, C2-C10 substituted alkenyl, C2-C11 substituted alkenyl or C2-C12 substituted alkenyl. Here, for example, C2-C10 alkyl means linear or branched alkenyl containing 2 to 10 carbon atoms, vinyl (CH ₂ ═CH—), allyl (CH ₂ ═CHCH ₂ —), CH ₃ CH═CH— and the like are exemplified. Also, for example, C2-C10 substituted alkenyl refers to C2-C10 alkenyl, in which one or more hydrogen atoms are substituted with substituents.

  As used herein, “optionally substituted alkenyl” means that either “alkenyl” or “substituted alkenyl” as defined above may be used.

As used herein, “alkenylene” refers to a divalent group formed by losing two hydrogen atoms from an aliphatic hydrocarbon having one double bond in the molecule, and is generally —C _n H _2n-2 —. (Where n is a positive integer greater than or equal to 2). “Substituted alkenylene” refers to alkenylene in which H of alkenylene is substituted by the substituent specified below. Specific examples include C2-C25 alkenylene or C2-C25 substituted alkenylene, especially C2-C3 alkenylene, C2-C4 alkenylene, C2-C5 alkenylene, C2-C6 alkenylene, C2-C7 alkenylene, C2 -C8 alkenylene, C2-C9 alkenylene, C2-C10 alkenylene, C2-C11 alkenylene or C2-C12 alkenylene, C2-C3-substituted alkenylene, C2-C4-substituted alkenylene, C2-C5-substituted alkenylene, C2- C6-substituted alkenylene, C2-C7 substituted alkenylene, C2-C8 substituted alkenylene, C2-C9 substituted alkenylene, C2-C10 substituted alkenylene, C2-C11 substituted alkenylene or C2~C12 substituted alkenylene are preferred. Here, for example, is C2~C10 alkyl means a straight or branched alkenylene including 2-10 carbon _{atoms, -CH = CH -, - CH} = CHCH 2 -, - (CH 3) C = CH- and the like are exemplified. Further, for example, C2-C10 substituted alkenylene refers to C2-C10 alkenylene, in which one or more hydrogen atoms are substituted with substituents. As used herein, “alkenylene” may contain one or more atoms selected from an oxygen atom and a sulfur atom.

  As used herein, “optionally substituted alkenylene” means that either “alkenylene” or “substituted alkenylene” as defined above may be used.

  As used herein, “cycloalkenyl” refers to alkenyl having a cyclic structure. “Substituted cycloalkenyl” refers to cycloalkenyl in which H of cycloalkenyl is substituted by the substituent specified below. Specific examples include C3-C4 cycloalkenyl, C3-C5 cycloalkenyl, C3-C6 cycloalkenyl, C3-C7 cycloalkenyl, C3-C8 cycloalkenyl, C3-C9 cycloalkenyl, C3-C10 cycloalkenyl, C3-C11. Cycloalkenyl, C3-C12 cycloalkenyl, C3-C4 substituted cycloalkenyl, C3-C5 substituted cycloalkenyl, C3-C6 substituted cycloalkenyl, C3-C7 substituted cycloalkenyl, C3-C8 substituted Cycloalkenyl, C3-C9 substituted cycloalkenyl, C3-C10 substituted cycloalkenyl, C3-C11 substituted cycloalkenyl or C3-C12 substituted cycloalkenyl. For example, preferable cycloalkenyl includes 1-cyclopentenyl, 2-cyclohexenyl and the like.

  In the present specification, “optionally substituted cycloalkenyl” means either “cycloalkenyl” or “substituted cycloalkenyl” as defined above.

As used herein, “alkynyl” refers to a monovalent group formed by losing one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule, such as acetylene, and is generally C _n H _{2n −3} − (where n is a positive integer of 2 or more). “Substituted alkynyl” refers to alkynyl in which H of alkynyl is substituted by the substituent specified below. Specific examples include C2-C3 alkynyl, C2-C4 alkynyl, C2-C5 alkynyl, C2-C6 alkynyl, C2-C7 alkynyl, C2-C8 alkynyl, C2-C9 alkynyl, C2-C10 alkynyl, C2-C11 alkynyl, C2-C12 alkynyl, C2-C3 substituted alkynyl, C2-C4 substituted alkynyl, C2-C5 substituted alkynyl, C2-C6 substituted alkynyl, C2-C7 substituted alkynyl, C2-C8 substituted Alkynyl, C2-C9 substituted alkynyl, C2-C10 substituted alkynyl, C2-C11 substituted alkynyl or C2-C12 substituted alkynyl. Here, for example, C2-C10 alkynyl means, for example, straight-chain or branched alkynyl containing 2 to 10 carbon atoms, and includes ethynyl (CH≡C-), 1-propynyl (CH ₃ C≡C -) Etc. are exemplified. Further, for example, C2-C10 substituted alkynyl refers to C2-C10 alkynyl, in which one or more hydrogen atoms are substituted with a substituent.

  As used herein, “optionally substituted alkynyl” means that either “alkynyl” or “substituted alkynyl” as defined above may be used.

As used herein, “alkoxy” refers to a monovalent group formed by loss of a hydrogen atom of a hydroxy group of an alcohol, and is generally represented by C _n H _{2n + 1} O— (where n is 1 or more). Is an integer). “Substituted alkoxy” refers to alkoxy in which H of alkoxy is substituted by a substituent specified below. Specific examples include C1-C2 alkoxy, C1-C3 alkoxy, C1-C4 alkoxy, C1-C5 alkoxy, C1-C6 alkoxy, C1-C7 alkoxy, C1-C8 alkoxy, C1-C9 alkoxy, C1-C10 alkoxy, C1-C11 alkoxy, C1-C12 alkoxy, C1-C2-substituted alkoxy, C1-C3-substituted alkoxy, C1-C4-substituted alkoxy, C1-C5-substituted alkoxy, C1-C6-substituted alkoxy, C1-C7 substituted alkoxy, C1-C8 substituted alkoxy, C1-C9 substituted alkoxy, C1-C10 substituted alkoxy, C1-C11 substituted alkoxy or C1-C12 substituted alkoxy . Here, for example, C1-C10 alkoxy means linear or branched alkoxy containing 1 to 10 carbon atoms, and includes methoxy (CH ₃ O—), ethoxy (C ₂ H ₅ O—), An example is n-propoxy (CH ₃ CH ₂ CH ₂ O—).

  In the present specification, “optionally substituted alkoxy” means either “alkoxy” or “substituted alkoxy” as defined above.

  As used herein, “heterocycle (group)” refers to a group having a cyclic structure including carbon and heteroatoms. Here, the hetero atom is selected from the group consisting of O, S and N, and may be the same or different, and may be contained in one or more than one. Heterocyclic groups can be aromatic or non-aromatic and can be monocyclic or polycyclic. The heterocyclic group may be substituted.

  In the present specification, the “optionally substituted heterocycle (group)” may be either the “heterocycle (group)” or “substituted heterocycle (group)” defined above. Means.

  As used herein, “alcohol” refers to an organic compound in which one or more hydrogen atoms of an aliphatic hydrocarbon are substituted with a hydroxyl group. In the present specification, it is also expressed as ROH. Here, R is an alkyl group. Preferably R may be C1-C6 alkyl. Examples of the alcohol include, but are not limited to, methanol, ethanol, 1-propanol, 2-propanol and the like.

  In this specification, the “carbocyclic group” is a group containing a cyclic structure containing only carbon, and the above-mentioned “cycloalkyl”, “substituted cycloalkyl”, “cycloalkenyl”, “substituted cyclo A group other than “alkenyl”. The carbocyclic group can be aromatic or non-aromatic and can be monocyclic or polycyclic. The “substituted carbocyclic group” refers to a carbocyclic group in which H of the carbocyclic group is substituted with a substituent specified below. Specific examples include C3-C4 carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10. Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3- C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, C3-C11 substituted carbocyclic group or C3- It can be a C12 substituted carbocyclic group. The carbocyclic group can also be a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group. Examples of the carbocyclic group include those in which one hydrogen atom is deleted from the phenyl group. Here, the hydrogen deletion position may be any position chemically possible, and may be on an aromatic ring or a non-aromatic ring.

  In the present specification, the “optionally substituted carbocyclic group” means that any of the above-defined “carbocyclic group” or “substituted carbocyclic group” may be used.

  As used herein, “heterocyclic group” refers to a group having a cyclic structure including carbon and heteroatoms. Here, the heteroatoms are selected from the group consisting of O, S and N, and may be the same or different, and may be contained in one or more than one. Heterocyclic groups can be aromatic or non-aromatic and can be monocyclic or polycyclic. The “substituted heterocyclic group” refers to a heterocyclic group in which H of the heterocyclic group is substituted with a substituent specified below. Specific examples include C3-C4 carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10. Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3- C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, C3-C11 substituted carbocyclic group or C3- One or more carbon atoms of the C12 substituted carbocyclic group may be substituted with a heteroatom. Heterocyclic groups can also be those in which one or more heteroatoms are substituted for the carbon atoms of a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group. Examples of the heterocyclic group include thienyl group, pyrrolyl group, furyl group, imidazolyl group, and pyridyl group. The hydrogen deletion position may be any chemically possible position, and may be on an aromatic ring or a non-aromatic ring.

  In the present specification, a carbocyclic group or heterocyclic group may be substituted with a divalent substituent in addition to being substituted with a monovalent substituent as defined below. Such divalent substitutions can be oxo substitutions (= O) or thioxo substitutions (= S).

  In this specification, “halogen” refers to a monovalent group of elements such as fluorine (F), chlorine (Cl), bromine (Br), and iodine (I) belonging to Group 7B of the periodic table.

  As used herein, “hydroxy” refers to a group represented by —OH. “Substituted hydroxy” refers to a hydroxy in which H is substituted with a substituent as defined below.

  In the present specification, the “thiol” is a group (mercapto group) in which an oxygen atom of a hydroxy group is substituted with a sulfur atom, and is represented by —SH. “Substituted thiol” refers to a group in which H of mercapto is substituted with a substituent defined below.

As used herein, “cyano” refers to a group represented by —CN. “Nitro” refers to the group represented by —NO ₂ . “Amino” refers to a group represented by —NH ₂ . “Substituted amino” refers to amino substituted with H as defined below.

  In this specification, “carboxy” refers to a group represented by —COOH. “Substituted carboxy” refers to a carboxy H substituted with a substituent as defined below.

  As used herein, “thiocarboxy” refers to a group in which an oxygen atom of a carboxy group is substituted with a sulfur atom, and may be represented by —C (═S) OH, —C (═O) SH, or —CSSH. “Substituted thiocarboxy” refers to thiocarboxy H substituted with the substituents defined below.

As used herein, “acyl” refers to a monovalent group formed by removing OH from a carboxylic acid. Representative examples of the acyl group include acetyl (CH ₃ CO—), benzoyl (C ₆ H ₅ CO—), and the like. “Substituted acyl” refers to an acyl hydrogen substituted with a substituent as defined below.

In this specification, “amide” is a group in which hydrogen of ammonia is substituted with an acid group (acyl group), and is preferably represented by —CONH ₂ . “Substituted amide” refers to a substituted amide.

  As used herein, “carbonyl” refers to a generic term for a substance including — (C═O) — which is a characteristic group of aldehyde and ketone. “Substituted carbonyl” means a carbonyl group substituted with a substituent selected below.

  In this specification, “thiocarbonyl” is a group in which an oxygen atom in carbonyl is substituted with a sulfur atom, and includes a characteristic group — (C═S) —. Thiocarbonyl includes thioketones and thioaldehydes. “Substituted thiocarbonyl” means thiocarbonyl substituted with a substituent selected below.

In this specification, “sulfonyl” refers to a generic term for a substance including —SO ₂ —, which is a characteristic group. “Substituted sulfonyl” means a sulfonyl substituted with a substituent selected below.

  As used herein, “sulfinyl” is a generic term for a substance including a characteristic group —SO—. “Substituted sulfinyl” means sulfinyl substituted with a substituent selected below.

  As used herein, “aryl” refers to a group formed by leaving one hydrogen atom bonded to an aromatic hydrocarbon ring, and is included in the present specification as a carbocyclic group.

  In the present specification, unless otherwise specified, substitution refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group. One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.

  In the present specification, unless otherwise specified, substitution refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group. One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.

  Examples of the substituent in the present invention include alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, thiol, cyano, nitro, amino, carboxy, carbamoyl, acyl, acylamino, Examples include, but are not limited to, thiocarboxy, amide, substituted carbonyl, substituted thiocarbonyl, substituted sulfonyl, or substituted sulfinyl. Such a substituent can be appropriately used in the present invention when designing an amino acid.

  Preferably, when a plurality of substituents are present, each may be independently a hydrogen atom or alkyl, but not all of the plurality of substituents are hydrogen atoms. More preferably, independently when there are multiple substituents, each may be independently selected from the group consisting of hydrogen and C1-C6 alkyl. The substituents may all have a substituent other than hydrogen, but preferably have at least one hydrogen, more preferably 2 to n (where n is the number of substituents) hydrogen. obtain. It may be preferred that the number of hydrogens in the substituent is large. This is because a large substituent group or a polar substituent group may have a hindrance to the effect of the present invention (particularly, interaction with an aldehyde group). Accordingly, the substituent other than hydrogen may preferably be C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, methyl and the like. However, since the effect of the present invention may be enhanced, it may be preferable to have a large substituent.

  In the present specification, C1, C2,... Cn represents the number of carbon atoms. Therefore, C1 is used to represent a substituent having 1 carbon.

  In this specification, “optical isomer” refers to one or a pair of a pair of compounds that have a mirror-image relationship with respect to a crystal or a molecular structure and cannot be superposed. It is a form of stereoisomer, and the other properties are the same, but only the optical rotation is different.

  As used herein, “protection reaction” refers to a reaction in which a protecting group such as Boc is added to a functional group desired to be protected. By protecting the functional group with the protecting group, the reaction of the functional group with higher reactivity can be suppressed, and only the functional group with lower reactivity can be reacted. The protection reaction can be performed by, for example, a dehydration reaction.

  As used herein, “deprotection reaction” refers to a reaction for removing a protecting group such as Boc. Examples of the deprotection reaction include a reaction such as a reduction reaction using Pd / C. The deprotection reaction can be performed, for example, by hydrolysis.

  In this specification, examples of the “protecting group” include a fluorenylmethoxycarbonyl (Fmoc) group, an acetyl group, a benzyl group, a benzoyl group, a t-butoxycarbonyl group, a t-butyldimethyl group, a silyl group, and a trimethylsilylethyl group. , N-phthalimidyl group, trimethylsilylethyloxycarbonyl group, 2-nitro-4,5-dimethoxybenzyl group, 2-nitro-4,5-dimethoxybenzyloxycarbonyl group, carbamate group and the like can be mentioned as typical protecting groups. . The protecting group can be used, for example, to protect a reactive functional group such as an amino group or a carboxyl group. Depending on the reaction conditions and purpose, various protecting groups can be used properly. An acetyl group, a benzyl group, a silyl group or a derivative thereof is used as a protective group for a hydroxy group, and a benzyloxycarbonyl group, a t-butoxycarbonyl group or a derivative thereof is used in addition to an acetyl group as a protective group for an amino group. can do. As the protective group for aminooxy group and N-alkylaminooxy group, trimethylsilylethyloxycarbonyl group, 2-nitro-4,5-dimethoxybenzyloxycarbonyl group or derivatives thereof are preferable.

  As used herein, “functional group that binds to cysteine” refers to a functional group that can be covalently bound to a cysteine residue side chain of a polypeptide. A typical functional group is a functional group that undergoes Michael addition reaction, and performs the following reaction.

Accordingly, the “moiety capable of covalently binding to a cysteine residue” includes any functional group capable of covalently binding to a cysteine residue by the mechanism as described above, and examples thereof include α, β unsaturated ketones. However, it is not limited to these.

  As used herein, “a moiety capable of interacting with an arginine residue or a tyrosine residue” includes, but is not limited to, a functional group that hydrogen bonds to a tyrosine residue. The “moiety capable of interacting with the tyrosine residue” includes a carbonyl group, a hydroxyl group, an amide group, and a thiol group (in the case of a substituent, a substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group). Can be a monovalent substituent containing groups), but is not limited thereto.

Examples of the compound represented by the general formula include, but are not limited to, the following compounds. The compounds of the present invention are, for example,
(Chemical Formula 8)
R ₁ —CH═CH— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —CH═CH—R ₂ —R ₃
Where R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 13 carbon atoms connecting the shortest distance, and R ₂ is A saturated or unsaturated hydrocarbon group which may be substituted, and R ₃ represents a substituent selected from the group consisting of hydrogen or an optionally substituted carbonyl group, hydroxyl group, amide group and thiol group; In the present invention, R ₂ and R ₃ may be a compound having a maximum number of carbon atoms connecting the shortest distance of 4 to 14 carbon atoms.

  Here, in this specification, “the maximum value of the number of carbons connecting the shortest distance”, when referring to a certain substituent, defines the shortest distance from any carbon atom contained in the substituent to another carbon atom. Is the maximum number of carbon atoms in a combination of all carbon atoms (the combination includes two carbons). For example, the benzene ring can be 6 × 5/2 combinations of carbons, and the shortest distance of the combination is either 1, 2 or 3. Therefore, the maximum value of the number of carbons connecting the shortest distance in this case is 3. The maximum value of the number of carbons connecting the shortest distance is approximately proportional to the standard of the region that can be occupied to the maximum for a certain substituent. Therefore, when designing a ligand, it can be understood that the maximum value of the number of carbons connecting the shortest distance can be used as an index for maintaining the correspondence relationship for the region where the corresponding receptor interacts.

Preferably, R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 9 carbon atoms connecting the shortest distance, and R ₂ is optionally substituted. A good saturated or unsaturated hydrocarbon group, wherein R ₃ is a monovalent hydrocarbon group containing a substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group and a thiol group, wherein R ₂ And R ₃ have a maximum number of carbon atoms connecting the shortest distances of 6 to 12 carbons included.

Here, preferably the structure is
R ₁ — (C═O) —CH═CH—R ₂ —R ₃ ,
In the formula, R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 9 carbon atoms connecting the shortest distance, and R ₂ is an optionally substituted saturated group. Or an unsaturated hydrocarbon group, and R ₃ is a monovalent substituent containing hydrogen or an optionally substituted substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group. Yes, where R ₂ and R ₃ are 6 to 14 carbon atoms having the maximum number of carbons connecting the shortest distances among the contained carbons,
The combination is

It may be preferable to have the carbon number in the right column for the nuclear receptor having the amino acid sequence shown in SEQ ID NO., But it is not limited thereto.

Specific compounds mentioned above, for example, such as indomethacin NSAIDs, 15-deoxy - [delta ^{12, 14} - prostaglandin _J 2 (11- oxo - prostaglandin -5Z, 9,12E, 14Z- tetraene - 1-Oic acid; 15d-PGJ ₂ ), 11-oxo-prosta-5Z, 12E, 14Z-triene-1-oic acid (CAY10410), 5-oxo-6E, 8Z, 11Z, 14Z-eicosatetraenoic acid (5-oxoODE), 9-oxo-10E, 12Z-octadecanodienoic acid (9-oxoODE), 13-oxo-9Z, 11E-octadecanodienoic acid (13-oxoODE) and 15-oxo -5Z, 8Z, 11Z, 13E-eicososatetraenoic acid (5-oxoETE) and the like. Not a constant.

  As used herein, “binding” between an agonist and a functional group that binds to a cysteine residue may be direct (ie, not via a linker) or indirectly (ie, via a linker). It may be.

As used herein, the term “linker” refers to a molecule that indirectly couples two different compounds (or parts of a compound). Exemplary linkers include, but are not limited to, any group that does not affect affinity, such as an alkyl chain. A method of designing a linker for separating a desired distance between an agonist and a functional group bonded to a cysteine residue is well known. For example, in order to calculate the distance between carbon atoms in an alkyl chain and produce a linker having a desired distance (Å), the maximum number of carbon atoms connecting the shortest distance according to the distance Alkyl chains having can be used.
Since the compound used in the present invention often has a structure similar to leukotriene, for example, Synthetic Organic Chemistry, Vol. 48, No. 7 (1990); Acharya et al., Tetrahedron Lett. 45 (2004), 1199- 1202, etc. It is understood that any synthetic procedure described in the review can be used.

  In each method of the present invention, the target product is a contaminant (unreacted weight loss, by-product, solvent, etc.) from the reaction solution, and a method commonly used in the art (for example, extraction, distillation, washing, concentration). , Precipitation, filtration, drying, etc.) followed by a combination of post-treatment methods commonly used in the art (eg, adsorption, elution, distillation, precipitation, precipitation, chromatography, etc.).

(Medical)
As used herein, “prophylaxis” or “prevention” refers to treating a disease or disorder so that such a condition does not occur before such a condition is triggered.

  As used herein, “treatment” refers to preventing a disease or disorder from deteriorating, preferably maintaining the status quo, more preferably reducing, when a certain disease or disorder becomes such a condition. Preferably, it means that it is reduced.

(screening)
As used herein, “modulation” refers to a change in biological activity when referring to a nuclear receptor.

  As used herein, “inhibition” refers to the reduction or elimination of biological activity when referring to a nuclear receptor.

  As used herein, “promotion” refers to the occurrence of a state from the state in which the biological activity is increased or absent when referring to a nuclear receptor.

  As used herein, “biological test” refers to determining whether a compound has a certain activity using an actual biological reaction. Biological tests include in vitro and in vivo tests. Biological testing is therefore an opposite concept to in silico.

  As used herein, a “candidate compound” refers to a candidate compound that can be used to modulate the activity of a target of interest (eg, a nuclear receptor) or to treat a target disease or disorder. . Thus, a compound can be referred to as a candidate compound if it is predicted to be effective for the disease or disorder of interest.

  As used herein, “compound species” refers to a single compound having a desired property, such as having a specific target activity in a certain group of compounds. For example, if a compound that modulates the activity of PPARγ is identified in a collection of compounds that modulate the activity of PPARγ (eg, has an agonistic effect), such a compound may be referred to as a compound species. In this specification, it is also simply referred to as a compound.

  As used herein, “library” refers to a certain collection of compounds for screening. The library may be a collection of compounds having similar properties or a collection of random compounds. Preferably, a set of compounds predicted to have similar properties is used, but is not limited thereto. The compound library used in the present invention can be prepared or obtained by any means including, but not limited to, combinatorial chemistry techniques, fermentation methods, plant and cell extraction procedures, and the like. it can. Methods for creating combinatorial libraries are well known in the art. For example, E.I. R. Felder, Chimia 1994, 48, 512-541; Gallop et al. Med. Chem. 1994, 37, 1233-1251; A. Houghten, Trends Genet. 1993, 9, 235-239; Houghten et al., Nature 1991, 354, 84-86; Lam et al., Nature 1991, 354, 82-84; Carell et al., Chem. Biol. 1995, 3, 171-183; Madden et al., Perspectives in Drug Discovery and Design 2, 269-282; Cwirla et al., Biochemistry 1990, 87, 6378-6382; Brenner et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383; Gordon et al. Med. Chem. 1994, 37, 1385-1401; Lebl et al., Biopolymers 1995, 37 177-198; and references cited therein. These references are incorporated herein by reference in their entirety.

  As used herein, “interaction” refers to an action between one molecule and another molecule when two or more molecules exist. Such interactions include hydrogen bonding, van der Waals forces, ionic interactions, nonionic interactions, receptor ligand interactions, electrostatic interactions and host-guest interactions. It is not limited. In the screening of the present invention, in particular, a covalent bond and / or a hydrogen bond can be used.

  In this specification, “covalent bond” refers to a chemical bond formed by an electron pair shared by two atoms. Whether a bond is a covalent bond can be identified by determining that the bond energy between two atoms is 50-1000 kJ / mol (preferably 60-700 kJ / mol).

  In the present specification, the term “hydrogen bond” refers to a hydrogen atom covalently bonded to an atom X having a high electronegativity (eg, F, O, Cl, etc.) and an atom Y having a high electronegativity (eg, F, It may be the same as O, Cl, and X), and represents a non-covalent bond in the form of X—H. In this specification, the bond energy of hydrogen bonds is usually about 8 to 35 J, and can be identified by determining the bond energy.

  As used herein, “evaluation”, when used in screening, refers to determining whether a candidate compound meets the requirements of such an indicator with respect to an indicator (eg, pharmacological activity). Such evaluation can be performed using methods known in the art and can be performed in silico (using a computer) or wet (performing an actual biological assay). For example, in the case of PPARγ, it can be carried out by checking whether the PPARγ activity has changed using its ligand or the like.

In the present specification, the “three-dimensional structure (computer) model” of a protein refers to a three-dimensional structure model of a certain compound expressed using a computer. Such a model can be displayed using a computer program known in the art, and examples of such a program include a program supported by CCP4, DENZO (HKL2000), MolScript (Avatar Software AB), Raster3D, PyMOL (DeLano Scientific), TURBO-FRODO (AFMB-CNRS), O (A. Jones, Uppsala University)
tet, Sweden), ImageMagic (John Chrysty), RasMol (University of Massachusetts, Amherst MA USA), and the like. Such a program can generate a model using, for example, atomic coordinate data as shown in FIG.

  In this specification, the “data array” indicates a series of data such as a numerical sequence indicating atomic coordinates.

  In the present specification, the “recording medium” can be any medium as long as data can be recorded. Examples of such a medium include a hard disk, MO, CD-R, CD-RW, CD-ROM, DVD-RAM, DVD-R, DVD-RW, DVD + RW, DVD-ROM, and memory card. It is not limited to them.

  In this specification, the “transmission medium” can be any medium as long as it can transmit data. Examples of such a medium include, but are not limited to, the Internet, an intranet, a LAN, and a WAN.

  As used herein, “pharmacophore” refers to a combination of atoms and functional groups (and their three-dimensional positions) that allows a drug to interact with a target protein in a specific manner. And shows its pharmacological activity. A three-dimensional “functional form” formed by the steric (physical) and electric fields of a drug molecule, which results in the pharmacological activity of the molecule. Various approaches have been developed to study measurable activity of pharmaceutical lead compounds and lead compounds against specific targets, and a pharmacophore can be designed from a series of structure-activity relationships.

  As used herein, “binding pocket” refers to a region of a molecule or molecular complex that associates with other chemicals or compounds as a result of its shape.

  As used herein, “active pocket”, when referring to a protein, refers to a region of a molecule or molecular complex that includes a site that can interact with its substrate or coenzyme.

  In the present specification, the “active site binding pocket” of a nuclear receptor (for example, PPARγ) is an active site binding pocket of a ligand-conjugated nuclear receptor because its shape binds to a general ligand. Or a part of a molecular complex that is similar to all or any part of The commonality of this shape is, for example, less than 3.0 mm, preferably less than 2.5 mm from the structural coordinates of the skeletal atoms of the amino acids constituting the binding pocket in the ligand-conjugated PPARγ (see FIGS. 17 and 18). Defined by root mean square deviation. The method for obtaining this calculation is as described elsewhere herein.

  The “active site binding pocket” or “active site” of ligand-conjugated PPARγ means the region of PPARγ that bears the binding region of the ligand.

  In the present specification, “structural coordinates” refer to orthogonal coordinates derived from mathematical expressions related to a pattern obtained by diffraction of a monochromatic beam of X-rays by an atom (scattering center) of a nuclear receptor such as PPARγ or a complex thereof. The diffraction data is used to calculate an electron density map of the crystal repeat unit. The electron density map is then used to determine the position of individual atoms in a nuclear receptor such as PPARγ.

  Such structural coordinate “data” (also referred to as atomic coordinate data) typically includes atomic coordinate data, topology, and molecular force field constants. The atomic coordinate data is typically data obtained from X-ray crystal structure analysis or NMR structure analysis, and such atomic coordinate data is obtained by newly performing X-ray crystal structure analysis or NMR structure analysis. Or can be obtained from a known database (eg, Protein Data Bank (PDB)). Atomic coordinate data can also be data created by modeling or calculation.

  The topology can be calculated using a commercially available or freeware tool program, but a self-made program may also be used. In addition, a molecular topology calculation program attached to a commercially available molecular force field calculation program (for example, prepar program attached to PRESTO, Protein Engineering Laboratory, Inc.) can be used. The molecular force field constant (or molecular force field potential) can also be calculated using a commercially available or freeware tool program, but self-made data may also be used. Further, molecular force field constant data attached to a commercially available molecular force field calculation program (for example, AMBER, Oxford Molecular) can be used.

  One skilled in the art readily understands that the set of structural coordinates for a nuclear receptor or nuclear receptor complex or part thereof, such as PPAR, is a set of relative points that define a shape in three dimensions. To do. Thus, different sets of coordinates can define similar or identical shapes, and slight changes in individual coordinates have little effect on the overall shape. With regard to binding pockets, these changes are not expected to significantly change the nature of the ligand that can associate with these pockets.

  Changes in the above coordinates can be generated by mathematical manipulation of PPARγ structural coordinates. For example, structural coordinates can be manipulated by crystallographic substitution of structural coordinates, division of structural coordinates, integer addition or subtraction to a set of structural coordinates, or any combination of the above.

(Crystal structure analysis)
Macromolecular structures (eg, polypeptide structures, or structures of candidate compounds) can be described in terms of various levels of configuration. For a general discussion of this configuration, see, for example, Alberts et al., Molecular Biology of the Cell (3rd edition, 1994), and Canter and Schimmel, Biophysical Chemistry Part I: The Conformation of Biologic 19 (see References). “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally arranged three-dimensional structures within a polypeptide. These structures are sometimes referred to herein as domains.

  In this specification, “crystal structure analysis” refers to analysis of a crystal structure using diffraction by an X-ray, electron beam, or neutron beam crystal. While applying an almost monochromatic and parallel line bundle to a single crystal, the orientation of the crystal is systematically changed, and the Bragg reflection intensity from many crystal network planes is recorded on a film or imaging plate with a Weissenberg camera or a precession camera. There is a method of measuring the reflection intensity systematically with a four-axis diffractometer equipped with a counter tube. From the reflection extinction law thus obtained, the space group to which it belongs can be determined. The presence or absence of a center of symmetry can also be determined from the statistics of integrated reflection intensity. After obtaining information such as the space group and the center of symmetry, the number of chemical units (number of atoms or molecules) contained in the unit cell can be determined from the molecular formula of the crystal and the size of the unit cell. In the heavy atom method, the Patterson function is used to find the position of the heavy atom (if it exists), and the contribution is analyzed as a clue. Even when heavy atoms are not included, the structure can be solved by isomorphous substitution using a heavy atom as an exogenous input. In complex protein molecules, by putting heavy atoms such as mercury in one position, especially by putting another kind of heavy atom in another position, a considerable number of reflection phases can be determined. Based on the analysis of the structure.

  Such a crystal structure analysis of a substance such as a protein can be performed using a method well known in the art. Such methods are described in, for example, X-ray crystal structure analysis of proteins (Springer Fairlark Tokyo), introduction to crystal analysis for life science (Maruzen), etc. Can be used arbitrarily.

  In general, it is known that protein crystals are considerably damaged by X-rays. Therefore, in order to succeed in X-ray crystal structure analysis, it is important to obtain crystals that are not easily damaged. In recent years, it is often performed to obtain high quality and high resolution diffraction data by freezing crystals and measuring diffraction data in a frozen state (Methods in ENZYMOLOGY Vol. 276, Macromolecular Crystallography, Part). A, C. W. Carter, Jr. and RM Sweet, Practical Crystallography (DW Rodgers)). In general, protein crystals are frozen by a method such as treatment with a solution containing a freeze stabilizer such as glycerol for the purpose of preventing the crystals from being collapsed by freezing. Frozen crystals can also be prepared by performing the instant freezing operation on crystals immersed in a storage solution to which a freeze stabilizer is added.

  In the present specification, heavy atom isomorphous substitution method (Methods in ENZYMOLOGY Vol. 115, Diffractipn Methods for Biological Macromolecules, Part B, HW Wyckoff, C. H. W. Hirs, and S.N. Methods in ENZYMOLOGY vol. 276, Macromolecular Crystallography, Part A, CW Carter, Jr. and RM Sweet edition) or multi-wavelength anomalous dispersion method (S. N. Timasheff edition, and Method YMO , Macromolecular Crystallography, Part , C.W.Carter, Jr., And R.M.Sweet eds) also it is possible to perform structural analysis by using. Here, the three-dimensional structure is obtained by obtaining the initial phase for calculating the electron density from the diffraction intensity difference between the diffraction data of the native crystal and the heavy atom derivative crystal, or the diffraction intensity difference between the diffraction data measured at different wavelengths. Can be determined. In determining the three-dimensional structure of a nuclear receptor (for example, PPARγ) to which the heavy atom isomorphous substitution method or multiwavelength anomalous dispersion method is applied, heavy atoms containing, for example, mercury, gold, platinum, uranium, selenium atoms, etc. as heavy metal atoms Derivative crystals can be used. For example, a heavy atom derivative crystal obtained by an immersion method using a mercury compound EMTS or potassium dicyanogold (I) is used.

  Diffraction data of native crystals and heavy atom derivative crystals can be measured using, for example, a protein crystal structure analysis beam line of R-AXIS IIc (Rigaku Denki) or SPring-8 (Nishiharima Large Synchrotron Radiation Facility). Diffraction data measurement at a plurality of wavelengths for applying the multi-wavelength anomalous dispersion method can be performed using, for example, a SPring-8 protein crystal structure analysis beam line. The measured diffraction image data is processed into reflection intensity data using, for example, a data processing program attached to R-AXIS IIc or a program DENZO (Mac Science) or a similar image processing program (or single crystal analysis software). . From the reflection intensity data of native crystals, the reflection intensity data of heavy atom derivative crystals at multiple wavelengths, the position of heavy metal atoms bound to proteins in the crystal using the difference Patterson diagram After obtaining, for example, using the program PHASES (W.Furey, University of Pennsylvania or CCP4 (British Biotechnology & Biological Science Research Counil, SERC) or the same diffraction data analysis program using the same diffraction data analysis program using the same diffraction data analysis program) The determined initial phase is, for example, the program DM (CCP4 package) or a similar phase improvement program (electron density modification). In accordance with the solvent smoothing method and the histogram matching method using a good program), the solvent region in the PPARγ ligand-binding fragment crystal is set to 30-50%, for example, and phase extension calculation is performed gradually from low resolution to high resolution. In the present specification, 30-60% of the volume of the protein crystal is occupied by solvent molecules other than protein (mainly water molecules). The volume is defined as the “solvent region.” Generally, when the obtained protein crystal has non-crystallographic symmetry, it is performed by averaging electron density called non-crystallographic symmetry (NCS) averaging. It is possible to further improve the reliability of the phase.Electron density diagram and refinement calculated using the phase after applying the solvent smoothing method Non-crystallographic symmetry matrix including translation and rotation can be calculated from the heavy atom coordinates, and at the same time, the region where protein molecules called masks exist can be identified from the electron density map obtained by the solvent smoothing method. By using the non-crystallographic symmetry matrix and the mask, NCS averaging calculation is performed by a program DM or the like, so that the phase is improved to a highly reliable phase and an electron density diagram used for constructing a three-dimensional structure model is obtained.

  A three-dimensional structural model of a nuclear receptor (eg, PPARγ) or a nuclear receptor ligand-binding fragment was displayed on three-dimensional graphics by, for example, the program O (O) (A. Jones, Uppsala Universitet, Sweden), etc. From the electron density diagram, it can be constructed by the following procedure. First, a plurality of regions having a characteristic amino acid sequence (for example, a partial sequence containing a tryptophan residue) are searched for on an electron density map. Next, a partial structure of amino acid residues suitable for the electron density is constructed on the three-dimensional graphics using the program O while referring to the amino acid sequence starting from the found region. By sequentially repeating this operation, all amino acid residues of the nuclear receptor (eg, PPARγ) or nuclear receptor ligand-binding fragment are adapted to the corresponding electron density, and an initial three-dimensional structure model of the whole molecule is constructed. To do. The constructed three-dimensional structure model is used as a starting model structure, and the three-dimensional coordinates describing the three-dimensional structure are refined according to the refinement protocol of the structure refinement program XPLOR (AT Brunger, Yale University). The Also, a nuclear receptor (eg, PPARγ) or a nuclear receptor ligand-binding fragment (eg, a nuclear receptor (eg, PPARγ) comprising the amino acid sequence shown in SEQ ID NOs: 2, 4, 6,... 46 or The native crystal conformation of the nuclear receptor ligand binding fragment) and the nuclear receptor (eg, PPARγ) or nuclear receptor ligand binding fragment (eg, amino acids shown in SEQ ID NOs: 2, 4, 6 .... 46) Nuclear receptor containing sequence (eg, PPARγ) or modified nuclear receptor ligand binding fragment) The native crystal conformation is determined by molecular replacement using the resulting EMTS derivative crystal conformation. Each three-dimensional structure can be determined by determining and following the above-described electron density improvement, model construction, and structure refinement procedures. This completes the determination of the conformation of the nuclear receptor (eg, PPARγ) or nuclear receptor ligand binding fragment of the present invention. The three-dimensional structure of the determined nuclear receptor (eg, PPARγ) or nuclear receptor ligand-binding fragment is registered in the protein data bank (PDB), which is an official data bank of protein three-dimensional structure, from the viewpoint of topology and the like. A comparison can be made with the conformation of different proteins (including fragments of proteins that are similar in function to PPARγ). In making the nuclear receptors (eg, PPARγ) or nuclear receptor ligand binding fragments of the present invention, topologies such as α helices, β strands, β sheets, etc. may be considered.

  Therefore, in the present invention, it is also intended to provide a drug (for example, an inhibitor, an activator, etc.) by computer modeling based on the disclosure of the present invention.

(NMR)
In this specification, “NMR” means nuclear magnetic resonance. When a group of nuclei having a magnetic moment is placed in the static magnetic field B ₀ , the nuclear Zeeman effect distributes to discontinuous energy levels according to the magnitude of the magnetic moment and the strength of the magnetic field. The phenomenon in which resonance absorption is observed when an electromagnetic wave having a frequency corresponding to this level interval is irradiated is called nuclear magnetic resonance.

  In this specification, “two-dimensional NMR” refers to a method of developing a nuclear magnetic resonance spectrum on two frequency axes. A plurality of pulses are used for this measurement, and Fourier transform is performed with the time interval and the time after the observation pulse as two time axes. The signal intensity is usually represented by contour lines. Typically, as two-dimensional NMR, TROSY (transverse relaxation-optimized spectroscopy), COSY (two-dimensional shift correlation NMR method), SECSY (two-dimensional spin echo correlation spectroscopy), FOCSY (two-dimensional folding correction spectroscopy) , HOHAHA (two-dimensional homonuclear Hartman method), NOESY (two-dimensional NOE method), 2D-J method (two-dimensional J-resolved spectroscopy), and relay COSY (relay coherence transfer spectroscopy), but are not limited thereto. . A preferred NMR is TROSY. TROSY is a method suitable for ultra-high magnetic field NMR developed by Wutrich et al. (Proc. Natl. Acad. Sci. USA vol. 94, 12366-12371 (1997)). In NMR of a large molecular weight molecule, the nuclear magnetic relaxation dominates (1) magnetic dipole interaction and (2) chemical shift anisotropy. Becomes so large that the relaxation time is shortened and the line width is increased. TROSY is a method of extending the relaxation time and narrowing the line width by canceling these two interactions. TROSY has made it possible to obtain two-dimensional NMR spectra for high molecular weight proteins (eg, 900 kDa or more).

In the present specification, when performing two-dimensional NMR, stable isotope nuclides used for labeling proteins include, but are not limited to, ¹⁵ N, ¹³ C, ² H, or a combination thereof. As the types of stable isotope nuclei used for preferred labeling, ¹⁵ N single label, ¹³ C single label, ¹⁵ N / ² H double label, ¹³ C / ² H double label, ¹⁵ N / ¹³ C / ² H triple label It is a sign.

When NMR is used in the present invention, the amount of change in the axial direction of the NMR signal can be used. Such variation is the frequency axis of the two-dimensional NMR deployed for stable isotopes, is defined as the molecular orientation dependent variation in the axial direction, for example, when using a ^{1 H, 1 H-axis} direction For example, when ¹³ C is used, the amount of change dependent on the molecular orientation in the ¹³ C-axis direction can be used.

The protein sample used for higher-order structure analysis by NMR used in this specification is usually subjected to structural analysis by labeling with stable isotope by biosynthesis. In general, the target protein expression system is first established by genetically engineered Escherichia coli, etc., and a stable isotope-labeled carbon source or nitrogen source (glucose with ¹³ C labeling of all carbon, ammonium chloride with ¹⁵ N labeling of nitrogen, etc.) By expressing in a medium supplemented with a protein, all carbon and nitrogen stable isotope-labeled proteins can be obtained. The obtained labeled protein can be subjected to NMR measurement by being purified by chromatography or the like and then concentrated by ultrafiltration or the like. At this time, it is desirable to pay attention to the following conditions.

  As for the container to be used, it is preferable to apply a coating treatment with silicon or the like to the equipment to be used including the NMR sample tube in order to suppress adhesion and dispersion to the glass wall surface in consideration of the high viscosity of the protein. Further, it is desirable to use a Pasteur pipette or the like for handling the sample.

  As the solvent, an aqueous solution is usually used. However, protein has many exchangeable amide protons, and if it is deuterated, a lot of information will be lost due to deuterium substitution of the key amide proton in the analysis of the spectrum. Usually, a sample is prepared with a light aqueous solution, and then about 10% heavy water is added for NMR lock. If necessary, a surfactant, a reducing agent, or the like may be added.

  The sample concentration is usually about 1 mM (for example, 0.4 mM to 2 mM). If there is a sufficient amount of sample and the solubility is high, measurement is possible even at 5 mM to 10 mM, but it should be noted that if the concentration is increased, there is a risk of association in the solution. In order to prevent an unexpected analysis result due to the association, it is preferable to confirm the presence or absence of the association by comparing the linearity between the low and high concentration samples.

  The pH to be used is usually preferably about 5 to 7, but is not limited thereto. This is because lowering the pH slows the exchange rate of exchangeable protons such as amide protons. However, if the pH is low, there is a concern of affecting the higher-order structure, so it is preferable to confirm with a circular dichroism (CD) spectrum or the like.

  As the buffering agent, it is desirable to use a buffer that does not have a proton in order to prevent an obstacle to spectrum analysis. Examples of such include, but are not limited to, phosphate buffer and deuterated acetic acid buffer.

  The temperature to be used is preferably about 30 to 40 ° C. in order to approach the temperature inside the living body. By setting the temperature higher and lowering the viscosity of the solution, the T2 of the protein molecule becomes longer, so that generally a better spectrum is obtained at a higher temperature.

  If dissolved oxygen is contained in the sample solution, the relaxation time is shortened due to paramagnetic relaxation, resulting in loss of sensitivity, which may hinder the detection of TROSY correlation. It is preferable to take care and remove dissolved oxygen. The degassing operation includes degassing by depressurization, bubbling of an inert gas, etc., and depressurized degassing is preferably used. Also, it is desirable not to generate bubbles.

  It is desirable to add azide for storage of the solution.

After sample preparation, NMR shim adjustment is performed. First, the resolution is adjusted by applying NMR lock. This is because protein solution NMR measurement is, as a rule, performed in a light aqueous solution, and therefore it is preferable to eliminate a huge water signal ( ¹ H concentration of tens of thousands to several million times that of the target protein). This is for the purpose of adjusting the resolution. Preferably, it is desirable to perform sufficient resolution adjustment for each measurement sample in order to obtain a good-quality spectrum. Since the sample tube is not normally rotated in protein solution NMR measurement, it is necessary to increase the resolution firmly, including adjustment of not only the Z axis but also the X, Y axis and higher order terms to obtain a good spectrum. Is necessary for.

  Next, the pulse width is measured. In protein solution NMR measured with an aqueous solution, the pulse width may vary depending on the sample due to the difference in salt strength. Therefore, in principle, it is necessary to measure the pulse width for each measurement sample.

  In the pulse sequence used in protein solution NMR, since a large number of RF pulses are arranged, it is considered that the deviation of the pulse width has a considerable influence on the whole. Therefore, it is desirable to measure the pulse width with the signal of the target protein and perform the multidimensional NMR measurement using the value before performing the multidimensional NMR measurement.

  Next, the sample is confirmed by NMR. It is desirable to perform NMR confirmation when there is a possibility that the protein has deteriorated, such as after storage for a while after the sample is prepared.

Therefore, perform a normal ¹⁵ N- ¹ H HSQC measurement, confirmed by two-dimensional NMR spectra. When there is a problem such as when the signal pattern is strange or when the phase does not match, the target protein may be broken, so it is necessary to adjust the experimental conditions again.

Next, spectrum analysis (signal attribution) is performed. The outline of the spectrum analysis method will be described below by taking the NOE spectrum as an example. The ¹ H signal can be assigned based on the distance information derived from NOE obtained by NMR, and a higher order structure can be derived. Overlapping signals are separated using measurements such as 3D and 4D, and assigned to known amino acid sequences. The principle is based on the fact that the spectrum has a specific pattern for each amino acid and that there are very characteristic laws such as chemical shift and spin coupling constant. Because of these characteristic properties, attribution is now automated. The characteristic chemical shifts of protein NMR spectra are shown below.

The ¹ H chemical shift of a protein is roughly limited by its environment. That is, a signal derived from an amide proton (near 8 ppm), a signal derived from a side chain aromatic ring (near 7 ppm), a signal derived from a proton at the α position (near 4 ppm), a signal derived from a proton at the β position (near 2 to 3 ppm) A signal derived from a methyl group (around 1 ppm) is observed.

^{13 C} have a similar correlation to chemical shifts, by selectively energizing each at ^{13 C} in the region, the carbon of the α-position or β-position, by handling as if it were another species and carbonyl carbon, It is used in various measurement methods for attribution.

  In addition to the assignment of amino acid sequences, changes in chemical shifts derived from higher order structures are seen. For example, a signal derived from the α-position proton is shifted in a high magnetic field in the α helix structure and is shifted in a low magnetic field in the β sheet structure. In addition, there may be a shift of about ± 1 ppm depending on the positional relationship with the side chain aromatic ring. In metal binding proteins and the like, a considerably large shift is observed at the site where the metal is bound.

Next, homonuclear spin coupling should also be considered. Since the amino acids constituting the protein are linked by peptide bonds (amide bonds), the spin bond between ¹ H- ¹ H is broken by the carbonyl group, and the spin bond between ¹ H- ¹ H is the same amino acid residue. Limited within group. Therefore, the pattern of spin coupling between ¹ H and ¹ H is characteristic for each type of amino acid, and amino acids are identified using this fact. Further, the dihedral angle φ derived from the ³ JH _α H _N spin coupling constant of the α-position proton and the amide proton is useful for determining the secondary structure of the main chain.

Also, heteronuclear spin coupling should be considered. The spin coupling constants other than ¹ H- ¹ H associated with the main chain, for example, H-N (90-100Hz), N-Cα (11Hz), Cα-H (140Hz), Cα-Cβ (30-40Hz ), Cα-C (═O) (55 Hz), Cα-XN (7 Hz), ¹³ C- ¹⁵ N (15 Hz), and the like.

NOE correlation should also be considered. The NOE correlation is used for higher-order structure analysis. The ¹ H homonuclear NOE sandwiched between the carbonyl group of the amide proton and the α-position proton has two amino acids that are adjacent to each other via an amide bond. It is derived from the group and is used in the assignment of amino acid sequence by ¹ H homonuclear experiment. This method is called sequence-specific linkage assignment method together with amino acid type information determined based on spin coupling and chemical shift.

  After the attribution work is completed, higher-order structural analysis is performed as necessary. Protein higher-order structures include α-helix structure in which amino acid sequence is formed with three-dimensional regularity, secondary structure such as β-sheet structure, and tertiary structure in which several secondary structures are spatially arranged. There is a quaternary structure in which domains constructed with a tertiary structure are spatially arranged, and it is important in structural analysis in protein solution NMR measurement to clarify these, and variously performed depending on the purpose.

  When the primary sequence is a known amino acid sequence, how a known polypeptide chain is entangled to construct a protein is resolved using information obtained from NMR. In this case, a structural optimization calculation algorithm called a distance geometry method or a molecular dynamics method with constraints is used. In either case, this is a technique for deriving a higher order structure by collecting NOE signals as distance information obtained from NMR as long as they can be collected and giving them to a calculation program as parameters.

  In this specification, the "molecular dynamics method with constraints" is a molecular dynamics method (a method that is commonly used for simulating molecular motion). This is a method of optimizing the structure by adding the constraint condition of distance derived from NOE as potential. This method uses Cartesian coordinates as position information, and because there are many variables, a huge amount of calculation is required and the load on the computer is large. This shortens the calculation time and is frequently used.

  In this specification, the “simulated annealing method” is a method of molecular dynamics calculation. Unlike general molecular dynamics calculation, a parameter that contributes to a covalent bond is set weakly at the initial stage of calculation, and the system This is a technique in which molecular motion is intensified by rapidly increasing the temperature, and then the temperature of the system is gradually lowered to slow down the molecular motion, while strengthening each parameter to converge the structure. Thereby, the structure can be prevented from being settled to the apparent convergence due to the structural change being hindered by the contact of atoms.

  In the present specification, the “distance geometry method” refers to a method of deriving a structure based on distance information and a set of bond angles (dihedral angles) as spatial position information. Usually, the bond length and bond angle of the covalent bond are fixed to the standard values of general proteins, and the structural calculation proceeds with the dihedral angle as a variable. Several structures that converge in the direction satisfying the constraint condition of the distance derived from NOE are taken out, and the structure that seems to be most suitable is taken as the final structure. This method is fast in calculation because there are few variables, and the load on the computer is small. However, structural changes are limited by contact of atoms (covalent bonds cannot be slipped through), so it is difficult to converge to the true structure. It is used to obtain the initial structure such as the molecular dynamics method with constraints described below.

  As mentioned above, although the NMR technique which can be used in this specification was demonstrated, such a technique is well-known in the said field | area, for example, Kurt Wuthrich, Kyogoku Yoshimasa, Kobayashi Yuji translation, NMR of protein and nucleic acid: Two Structural analysis by dimensional NMR, Tokyo Chemical Doujin. (1991); Sattler, J .; Schleucher, C.I. Griesinger, Progr. NMR Spectrosc. 34, 93-158. (1999); Cavanagh, W.C. J. et al. Fairbrother, A.M. G. Palmer III, N.I. J. et al. Skelton, Protein NMR Spectroscopy: Principles and Practice, Academic Press. (1995); L. James and N.M. J. et al. Openheimer (eds.), Methods in Enzymology, Vol. 239, Academic Press. (1994); Yoji Arata, NMR of proteins: interpretation and evaluation of structural data, Kyoritsu Shuppan. (1996); Yoji Arata, NMR book, Maruzen. (2000); Yasuaki Nemoto, Takuya Yoshida, Yuji Kobayashi, Science and Industry, 69 (10), 419-425. (1995); Yasuaki Nemoto, Takuya Yoshida, Yuji Kobayashi, Science and Industry, 70 (2), 48-55. (1996) and the like, the contents of which are incorporated herein by reference.

(Mass spectrometry)
In the present invention, mass spectrometry can be used to measure bonds such as covalent bonds.

  In the present specification, “mass spectrometry” refers to an arbitrary method for analyzing atomic / molecular ions based on a difference in mass using electromagnetic interaction. Typically, mass spectrometry is roughly classified into two methods. The first is a method of analyzing the trajectories of ions at the same time, and the apparatus is called a mass spectrometer. The second is a method of measuring the difference in mass by the time difference of ion motion or the difference of resonance conditions, although the trajectories are the same, and the apparatus is called a mass spectrometer.

  Mass spectrometry methods are well known in the art, and examples include, but are not limited to, MALDI-TOF. Those skilled in the art can appropriately modify such methods and use them in the present invention.

  In the present specification, binding can be calculated using information on quantitative changes in the structure of a substance such as a protein as obtained by mass spectrometry. Information on quantitative changes in the structure of such substances as proteins can be displayed as information on the presence or absence of the sugar chain itself, or the entire molecule (eg, protein) containing the sugar chain is displayed as information. You can also Information on quantitative changes can also be expressed as, but not limited to, the ratio of protein to ligand ratios of interest. Examples of a unit indicating the amount (or level) of each sugar chain or a molecule containing a sugar chain constituting the information of such quantitative change include ng, μmol, signal intensity in mass spectrometry, signal intensity in spectroscopic analysis , Or mg / sample g, mmol / sample g, or relative fluorescence intensity, but is not limited thereto. In the present invention, it may be important to analyze (by interval differentiation or the like) changes in such quantitative changes in protein structure.

MALDI-TOF (MS), which is a representative mass analysis, is an abbreviation for Matrix Assisted Laser Deposition Ionization-Time-of-Flight (Mass Spectrometer). MALDI is a technique found by Tanaka et al. And developed by Hillenkamp et al. (Karas M., Hillenkamp, F., Anal. Chem. 1988, 60, 2299-2301). In this method, a sample and a matrix solution are mixed at a molar ratio of (10-2 to 5 × 10-4): 1, and then the mixed solution is dried on a target to be in a crystalline state. Large energy is given to the matrix by the pulse laser irradiation, and sample-derived ions such as (M + H) + and (M + Na) + are desorbed from the matrix-derived ions. Even if it is contaminated with a small amount of phosphate buffer, Tris buffer, guanidine, etc., it can be analyzed. MALDI-TOF (MS) measures mass based on time of flight using MALDI. When an ion is accelerated at a constant acceleration voltage V, the ion mass is m, the ion velocity is v, the ion charge number is z, the elementary charge is e, and the ion flight time is t. m / z is
m / z = 2eVt2 / L2
It can be expressed as For such MALDI-TOF measurement, Shimadzu / Kratos's KOMPACT MALDI II / III / IV and the like can be used. In the measurement, a pamphlet created by the manufacturer can be referred to. The irradiation energy of laser irradiation used at the time of measurement of MALDI-TOF is referred to as dissociation energy, and analysis such as covalent bonding can be performed using the dissociation energy.

  Such a mass spectrometer can be used with reference to a manual provided by the manufacturer. Specifically, a sample is dropped on a target probe attached to a mass spectrometer together with a matrix (for example, solid or liquid) and dried, and the mass is measured with an analyzer. The matrix provided by the manufacturer can be used, and examples thereof include, but are not limited to, dithranol, HABA, DHBA, IAA and the like.

  In mass spectrometry, ionization efficiency is increased by adding salts such as NaCl, KCl, NaTFA (trifluoroacetate), AgTFA as ionization aids (cation aids) as necessary to facilitate generation of proton adduct ions. It is also possible to raise. A person skilled in the art can select various conditions such as selection of a matrix and an ionization aid during structural analysis of each sugar chain.

(Transcriptional activity assay)
As used herein, “transcriptional activity assay” refers to any assay for testing whether a region has transcriptional activity. In this assay, the influence of a chemical substance on a series of actions that binds to a receptor in a cell and activates transcription from a gene of interest to a protein is evaluated. An example is illustrated in FIG. In FIG. 14, luciferase is illustrated as a protein, but it is not limited thereto. PPARγ is exemplified as the target gene. A GAL4 binding region is exemplified as a transcription factor recognition sequence upstream of the DNA encoding the reporter gene. Examples of transcriptional regulators include PPARγ binding domain (PPARγLBD) fused with GAL4 DNA binding domain (GAL4DBD). In this method, first, a cultured cell into which a DNA encoding a target nuclear receptor and a DNA encoding a reporter gene are introduced is prepared. A chemical substance is added to the culture medium containing the cultured cells and cultured, and the fluorescence intensity and the like are measured. Here, it is evaluated whether a series of actions from the binding to the receptor to the protein synthesis is similar to that of a hormone. In this method, it is possible to evaluate whether a chemical substance acts as an agonist or an antagonist by making a target hormone and a chemical substance coexist. Furthermore, in this method, it is possible to rapidly process a large amount of sample using a high-throughput apparatus that is a robot system.

  Accordingly, in the present invention, such a transcription activity assay can be used in carrying out a method for determining whether a candidate compound is an agonist or antagonist of a nuclear receptor. In this method, after providing a candidate compound, it is determined whether the candidate compound is covalently bound to cysteine in the nuclear receptor, the candidate compound determined to be covalently bound is selected, and the nuclear receptor is selected. A candidate compound in a system (eg, a cell) comprising a nucleic acid construct comprising a nucleic acid sequence encoding a reporter operably linked to a nucleic acid sequence comprising a region that interacts with said selected nuclear receptor Expose. Here, when the expression of the reporter is enhanced, the candidate compound is determined as an agonist of the nuclear receptor, and when the expression of the reporter decreases, the candidate compound is determined as an antagonist of the nuclear receptor.

  In the present specification, any reporter may be used as long as expression can be confirmed, but luciferase is typically used. It is because observation is easy because of chemiluminescence. In addition to this, for example, a fluorescent protein or the like can be used, but is not limited thereto.

  It will be appreciated that the system utilized in the transcriptional activity assay used in the present invention can be a cell, but is not limited thereto, and a cell-free system can also be used.

  Examples of the transcription factor recognition sequence include GAL4 DNA (Type CM, Klausing K., Methods Mol Med. 2003; 85: 175-83), LexA DBD (Chemistry and Biology, vol 10, no. 7, pp. 584-585 (Jury, 2003)) and the like, but is not limited thereto. It is understood that such a transcription factor recognition sequence can be used with any nuclear receptor. For other transcription factor recognition sequences, using this system of the present invention, a substance known to be an agonist was used in place of the candidate compound, and a transcription factor recognition sequence candidate sequence was used in place of the GAL4 DNA. It can be appreciated that any sequence in which the assay is run and a positive response by an agonist is seen in the assay can be used as a transcription factor recognition sequence.

(Computer modeling)
The present invention identifies, selects, and designs chemicals (including activators and inhibitory compounds) that can bind to nuclear receptors (eg, PPARγ (eg, binding pocket)) through the use of molecular design techniques. Make it possible for the first time.

  In one aspect, the present invention also provides a polypeptide comprising a binding pocket determined in the present invention based on information such as a ligand identified in the present invention.

  Our elucidation of ligand binding sites on nuclear receptors (eg PPARγ) designs novel chemicals and compounds that can interact with either or both of the nuclear receptor binding pockets Provide all or part of the information necessary for this purpose. This elucidation also allows the evaluation of structure activity data for analogs of other compounds that bind to the nuclear receptor-like binding pocket.

  In modeling, discussion of the ability of a substance to bind to, associate with, or inhibit a nuclear receptor-like binding pocket will only discuss the characteristics of the substance. Assays for determining whether a compound binds to a nuclear receptor are well known in the art and are described in, eg, Chen, M. et al. , Marumiya, S .; , Masaki, T .; , And Sawamura, T .; Biochem. J. et al. (2001) 355, 289-296; Chen. , M.M. Inoue, K .; Narumiya, S .; , Masaki, T .; , And Sawamura, T .; FEBS Lett. (2001) 499, 215-219; and Shi, X. et al. Niimi, S .; , Ohtani, T .; and Macida, S .; J. et al. Cell. Sci. (2001) 114, 1273-1282, etc., and the contents thereof are incorporated by reference in the present specification.

  The design of a compound that binds to or inhibits a nuclear receptor (eg, PPARγ) or a ligand-binding fragment thereof (eg, a binding pocket) according to the present invention generally involves consideration of two requirements. First, the material must be able to physically and structurally associate with some or all of the nuclear receptors. Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions.

  Second, the substance must be able to adopt a conformation that allows it to associate directly with the nuclear receptor or its ligand-binding fragment. Although certain parts of the substance are not directly involved in these associations, these parts of the substance can still affect the overall conformation of the molecule. This can then have a significant impact on efficacy. Such conformational requirements include the whole three-dimensional structure and orientation of chemicals associated with all or part of the binding pocket, or some of the direct interactions with nuclear receptors or their ligand-binding fragments or their homologues. Includes spatial arrangements between functional groups of substances including chemical substances.

  The potential inhibitory or binding effect of a chemical on a nuclear receptor (eg, PPARγ) or nuclear receptor ligand binding fragment can be analyzed by use of computer modeling techniques prior to its actual synthesis and testing. If the theoretical structure of a given substance suggests insufficient interaction and association between that substance and a nuclear receptor (eg, PPARγ) or nuclear receptor ligand binding fragment This test is excluded. However, if computer modeling shows a strong interaction, the molecule can then be synthesized and tested for its ability to bind to a nuclear receptor (eg, PPARγ) or nuclear receptor ligand binding fragment.

  Candidate compounds for modulators of nuclear receptors can be evaluated computationally by screening the chemical or fragment for their ability to associate with the nuclear receptor and selecting positive factors.

  One skilled in the art can use one of several methods for screening chemicals or fragments for the ability to associate with nuclear receptors or ligand binding fragments thereof. This process is based on, for example, PPARγ-like binding pockets on a computer screen, based on the structural coordinates of a PPARγ ligand-binding fragment, or variants or homologues thereof, or other coordinates that define similar shapes arising from computer-readable recording media. It can be initiated by visual inspection. The selected fragments or chemicals can then be placed in various orientations or docked within the binding pocket as defined above. Docking can be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields such as CHARMM and AMBER.

  Computer programs for performing computer modeling can also be used in the process of selecting fragments or chemicals. Examples of such programs include the following.

  1. GRID (P. J. Goodford, “A Computational Procedure for Determining Favorable Binding Sites on Biologically Implanted Macromolecules., 198-49. GRID is available from Oxford University, Oxford, UK.

  2. MCSS (A. Miraranker et al., “Functional Map of Binding Bindings: A Multiple Copy Simulaneous Method”) Proteins: Structure, Function 29, and 29, 29. MCSS is available from Molecular Simulations, San Diego, CA.

  3. AUTODOCK (DS Goodsell et al., “Automated Docking of Proteins to Proteins by Simulated Annealing”, Proteins: Structure, Function, and Genetics, p. 19, 19202). AUTODOCK is available from Scripts Research Institute, La Jolla, CA.

4). DOCK (ID Kuntz et al., “A Geometric Approach
to Macromolecule-Ligand Interactions, "J. Mol. Biol. 161, 269-288 (1982)). DOCK is available from the University of California, San Francisco, CA.

  Once the appropriate compound qualities or fragments (compound species) are selected, they can be assembled into a single compound or complex. Prior to construction, a visual representation of the relevance of each other fragment on a three-dimensional image displayed on a computer screen in relation to the structural coordinates of a nuclear receptor (eg PPARγ) or nuclear receptor ligand binding fragment An inspection can be performed. Following this, Quanta or Sybyl [Tripos Associates, St. Manual model building using software such as Louis, MO] is performed.

  Useful programs that can be used in linking individual chemicals or fragments include:

  1. CAVEAT (P.A.Bartlett et al.,.. "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules" (Molecular Recognition in Chemical and Biological Problems, Special Pub, Royal Chem.Soc, 78,182-196 (1989)); G, Lauri, and PA Bartlett, “CAVEEAT: a Program to Facility the Design of Organic Molecules”, J. Compute. Aided Mol. Des., 8, 51-66. 1994)). CAVEAT is available from the University of California, Berkeley, CA.

  2. A 3D database system such as ISIS (MDL Information Systems, San Leandro, CA). This field is C. Martin, “3D Database Searching in Drug Design”, J. Am. Med. Chem. 35, 2145-2154 (1992).

  3. HOOK (M.B. Eisen et al., “HOOK: A Program for Finding Novel Molecular Architecture, Saturity the Chemical. Sequential Refining of Bioc. (1994)). HOOK is available from Molecular Simulations, San Diego, CA.

  Instead of proceeding to build one fragment or chemical at a time as an inhibitor of PPARγ, etc. in a stepwise manner as described above, an inhibitory or other nuclear receptor (eg, PPARγ) or nuclear receptor ligand The binding compounds of the binding fragments can be designed globally or de novo, either using an empty binding site or optionally including some known inhibitor moiety. There are a number of new ligand design methods, including:

1. LUDI (H.-J. Bohm, "The Computer Program LUDI: A New Method for the De Novo Design."
of Enzyme Inhibitors, "J. Comp. Aid. Molec. Design, pp. 61-78 (1992)). LUDI is available from Molecular Simulations Incorporated, San Diego, CA.

2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, 8985 (1991)). LEGEND is a Molecular Simulations
Available from Incorporated, San Diego, CA.

  3. LeapFrog (available from Tripos Associates, St. Louis, MO).

  4). SPROUT (V. Gillet et al., “SPROUT: A Program for Structure Generation”, J. Comput. Aided Mol. Design, 7, 127-153 (1993)). SPROUT is available from University of Lees, UK.

Other molecular modeling techniques can also be used in accordance with the present invention [see, eg, N. C. Cohen et al., “Molecular Modeling Software and Methods for Medicinal Chemistry”, J. Am. Med. Chem. 33, 883-894 (1990); A. Navia and M.M. A. Murcko, “The Use of Structural Information in Drug Design”, Current Opinions in
See also Structural Biology, 2, 202-210 (1992); M.M. Balbes et al., “A Perspective of Modern Methods in Computer-Aided Drug Design,” Reviews in Computational Chemistry, vol. 1994)); C. Guida, “Software For Structure-Based Drug Design”, Curr. Opin. Struct. Biology. 4, 777-781 (1994)].

  Once a compound has been designed or selected by the methods described above, the efficiency by which the substance can bind to the binding pocket of a nuclear receptor (eg, PPARγ) or a nuclear receptor ligand binding fragment is evaluated by calculation. Can be tested and optimized. For example, an effective nuclear receptor (eg, PPARγ) or a binding pocket agonist of a nuclear receptor ligand-binding fragment preferably has a relatively small energy difference (ie, binding) between its bound and free states. Small deformation energy). Thus, the most efficient nuclear receptor (eg, PPARγ) agonist should preferably be designed with a deformation energy of binding of about 10 kcal / mole or less (more preferably 7 kcal / mole or less). . An agonist of a nuclear receptor (eg, PPARγ) can interact with the binding pocket in two or more conformations that are similar in total binding energy. In these cases, the deformation energy of binding is thought to be the difference between the energy of the free material and the average energy of these conformations observed when the agonist binds to the protein.

  A substance designed or selected to bind to a nuclear receptor (eg, PPARγ) or nuclear receptor ligand binding fragment is preferably electrostatically repulsive with its target enzyme and surrounding water molecules in its bound state. It can be further optimized by calculation so that there is no interaction. Such non-complementary electrostatic interactions include charge-charge repulsive interactions, dipole-dipole repulsive interactions and charge-dipole repulsive interactions.

Specific computer software is available in the field of assessing compound deformation energy and electrostatic interactions. Examples of programs designed for such use include: Gaussian 94, revision C (MJ Frisch, Gaussian, Inc., Pittsburgh, PA 1995); AMBER, version 4.1 ( P. A. Kollman, University of California at San Francisco, 1995); QUANTA / CHARMM (Molecular Simulations, Inc., San Diego, CA 1995); ); DelPhi (Molecular Simulations, Inc., S) n Diego, CA 1995); and AMSOL (Quantum Chemistry Program Exchange, Indiana University). These programs may be executed using, for example, a Silicon Graphics workstation (eg, Indigo ² with “IMPACT” graphics). Other hardware systems and software packages are also known to those skilled in the art.

  Another approach possible with the present invention is the computational screening of small molecule databases for chemicals or compounds that can bind in whole or in part to PPARγ. In this screening, the quality of such a substance's fit to the binding site can be determined by either geometric complementarity or estimated interaction energy [E. C. Meng et al. Comp. Chem. 16, 505-524 (1992)].

(Description of Preferred Embodiment)
The description of the preferred embodiment is described below, but it should be understood that this embodiment is an illustration of the present invention and that the scope of the present invention is not limited to such a preferred embodiment.

  In one aspect, the present invention is a method of identifying a compound that modulates a nuclear receptor, the method comprising: A) providing a candidate compound; and B) the candidate compound comprising the nuclear receptor Determining whether it binds covalently to cysteine in the body, wherein the candidate compound determined to be covalently bound is identified as a compound that modulates a nuclear receptor. provide.

  Here, the provision of the candidate compound can be performed using any technique known in the art. Examples of such include, but are not limited to, a method of synthesizing a compound library using combinatorial chemistry, a method of providing an existing compound library, and the like.

  Here, the technique for determining whether or not to covalently bind to cysteine in the nuclear receptor can be carried out by using the fluorescently labeled maleimide and the like provided in the present invention. Or it can also carry out using well-known techniques, such as a mass spectrometer or SDS-PAGE. The covalently bonded portion may be analyzed by crystal structure analysis or the like.

  In one embodiment, the cysteine targeted by the present invention is preferably present in the ligand binding domain, but is not limited thereto. The ligand binding domain, also referred to as the E domain, has its region determined or estimated at each nuclear receptor and is described in Table 7 herein. It is understood that even nuclear receptors other than those listed in this table have corresponding domains, such domains are based on Table 7 and Table 12, etc. It can be specified by identifying.

  In one embodiment, the nuclear receptor targeted by the present invention is PPARα, PPARβ / δ, PPARγ1, PPARγ2, ERR1, ERR2, ERR3, HNF4α, HNF4β, RARα, RARβ, RARγ1, RARγ2, RXRα, RXRβ, RXRγ, RORα, RORβ, RORγ, LRH1, SF1, TLX and O4245 receptors. In a particularly preferred embodiment, the nuclear receptor is PPARγ, but is not limited thereto.

  In one embodiment, the position of cysteine targeted by the present invention is the following table:

It is shown by the amino acid number in the SEQ ID NO shown.

In one embodiment, the candidate compound to be screened by the present invention may be any compound, but preferably R _A —CH═CH— (C═O) —R _B — or R _A — ( And a compound having a substituent of C═O) —CH═CH—R _B —. Here, in the formula, R _A is a monovalent substituent that includes hydrogen or an optionally substituted substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group, and R _B Is a divalent hydrocarbon group.

  In one embodiment, it is understood that any measurement method may be used for the measurement of the covalent bond, but it is preferably performed using the fluorescently labeled maleimide disclosed in the present invention. is there. Specific examples of such fluorescently labeled maleimide include, but are not limited to, rhodamine maleimide and Cy5-maleimide.

  In another embodiment, the present invention comprises the step of determining whether a candidate compound to which the present invention is directed hydrogen bonds with an amino acid in helix 12 or helix 5 in the ligand binding domain of said nuclear receptor. Includes.

  It is understood that the corresponding domain can be specified by alignment or the like as described in FIGS.

  In a preferred embodiment, the residue number of the amino acid in helix 12 or helix 5 is

It may be arginine or tyrosine represented by the amino acid number in SEQ ID NO.

  In another preferred embodiment, the amino acid in helix 12 or helix 5 targeted by the present invention comprises tyrosine 473 of the amino acid sequence shown in SEQ ID NO: 42 or tyrosine 501 of the amino acid sequence shown in SEQ ID NO: 44. Also good.

  In one embodiment, hydrogen bonds can be observed by means such as a nuclear magnetic resonance apparatus (NMR) or an X-ray diffractometer. The NMR and X-ray analyzers can be used using techniques known in the art. Specifically, the hydrogen bond can be measured as follows. It can be calculated by measuring the binding energy.

  In one embodiment, the covalent bond can be measured by a mass spectrometer or SDS-PAGE or the like. The mass spectrometer or SDS-PAGE can be used using techniques known in the art. Specifically, the covalent bond can be calculated, for example, by measuring the binding energy.

  In one embodiment, the regulatory function of the nuclear receptor to be screened can be inhibition or promotion. In the case of inhibition, the candidate compound is a candidate for an inhibitor (or inhibitor). When facilitating, the candidate compound can be a candidate for an activator (or activator). In the case of an inhibitor, the function of the target nuclear receptor is suppressed. Therefore, it can be a drug candidate when such suppression is desired. In the case of an activator, it activates the function of the target nuclear receptor. Therefore, it can be a drug candidate when such activation is desired.

In one embodiment, the present invention provides a compound identified by the present invention. This compound has the following formula:
(Chemical 9)
R ₁ —CH═CH— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —CH═CH—R ₂ —R ₃
In which R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 13 carbon atoms connecting the shortest distance, and R _{1 2} is a saturated or unsaturated hydrocarbon group that may be substituted, and R ₃ is selected from the group consisting of hydrogen or an optionally substituted carbonyl group, hydroxyl group, amide group, and thiol group It is a monovalent substituent containing a substituent. Here, R ₂ and R ₃ have a maximum number of carbon atoms connecting the shortest distances of 4 to 14 carbon atoms contained.

In a preferred embodiment, R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 9 carbon atoms connecting the shortest distance, and R ₂ is substituted A saturated or unsaturated hydrocarbon group, and R ₃ is a monovalent hydrocarbon group containing a substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group, For R ₂ and R ₃ , it is advantageous that the maximum number of carbon atoms connecting the shortest distance is 6 to 12 for the contained carbon. Although not wishing to be bound by theory, it is preferable that such a range is appropriate in the general structure of a nuclear receptor, considering the relative distance from the domain to interact with. It is understood that there is.

The present invention is also a composition for modulation of a nuclear receptor comprising a specified compound, wherein the compound has the formula:
(Chemical formula 10)
R ₁ —CH═CH— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —CH═CH—R ₂ —R ₃
Where R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 13 carbon atoms connecting the shortest distance, and R ₂ is A saturated or unsaturated hydrocarbon group which may be substituted, and R ₃ represents a substituent selected from the group consisting of hydrogen or an optionally substituted carbonyl group, hydroxyl group, amide group and thiol group; In the present invention, R ₂ and R ₃ provide a composition having a maximum number of carbon atoms connecting the shortest distance of 4 to 14 carbon atoms. This compound has been specified by the present invention. In particular, it was the first time in the present invention that an interaction with a nuclear receptor was known. Therefore, it can be said that it was first identified in the present invention that such a compound is an active ingredient as a nuclear receptor modulator.

In one specific embodiment, the structure is R ₁ — (C═O) —CH═CH—R ₂ —R ₃ , and in the compounds of the present invention, R ₁ is the number of carbons connecting the shortest distance An optionally substituted saturated or unsaturated hydrocarbon group having a maximum value of 1 to 9, R ₂ is an optionally substituted saturated or unsaturated hydrocarbon group, and R ₃ Is a monovalent hydrocarbon group containing a substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group and a thiol group, wherein R ₂ and R ₃ are the shortest distances between the contained carbons. The maximum number of carbon atoms to be connected is 6 to 12, and the combination is

To the nuclear receptor having the amino acid sequence shown in SEQ ID NO.

In one embodiment, the present invention provides a composition for the treatment, prevention or prognosis of a disease, disorder or condition resulting from a nuclear receptor. This compound has the following formula:
(Chemical Formula 11)
R ₁ —C═C— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —C═C—R ₂ —R ₃
Where R ₁ is a saturated or unsaturated hydrocarbon group having 1 to 13 carbon atoms, and R ₂ is a saturated or unsaturated carbon group having 4 to 14 carbon atoms. S ₃ is a saturated hydrocarbon group, and R ₃ is a monovalent substituent containing hydrogen or an optionally substituted substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group.

  Here, the disease, disorder or condition caused by the nuclear receptor refers to any disease, disorder or condition resulting from the function of the nuclear receptor, and examples thereof include various chronic diseases.

(Method for quantifying the binding ability of a ligand to a target biomolecule)
In one aspect, the present invention provides 1) a step of mixing a target biomolecule and a ligand to form a mixture; 2) a step of adding a fluorescent-labeled maleimide to the mixture; 3) in the mixture, the target biomolecule A method for quantifying the binding ability of a ligand to the target biomolecule, comprising the step of detecting the binding between the fluorescent label maleimide and the fluorescent label maleimide. Here, for mixing the target molecule and the ligand, any method known in the art can be used. Any fluorescent label maleimide may be used. For example, rhodamine maleimide may be used. Detection of the binding between the target biomolecule and the fluorescently labeled maleimide can be applied by applying any method known in the art, and for example, any method capable of measuring fluorescence or mass can be used. For example, as such a technique, a mass spectrometer or SDS-PAGE can be used, but is not limited thereto.

  In one preferred embodiment, in the method for quantifying the binding ability of a ligand to a target biomolecule of the present invention, the binding is a covalent bond, and further includes the step of quantifying the covalent bond, It can be achieved by measuring at least two quantities for the ligand. Here, quantification can be performed, for example, based on the signal intensity in the mass spectrometry or SDS-PAGE.

In a preferred embodiment, in the method for quantifying the binding ability of a ligand to a target biomolecule of the present invention, the quantification of the covalent bond is performed using an irreversibly covalently bound ligand as a probe. This embodiment is characterized in that the binding of non-covalent ligands is quantified. Examples of irreversibly covalently bonded ligands include NSAIDs such as indomethacin, 15-deoxy-Δ ^12,14 -prostaglandin J ₂ (11-oxo-prosta-5Z, 9, 12E, 14Z-tetraene-1 - oic acid; _15d-PGJ 2), 11- oxo - prostaglandin-5Z, 12E, 14Z-trien-1-oic acid (CAY10410), 9- oxo -10E, 12Z- octadecanoate dienoic acid (9-oxo ODE ), 13-oxo-9Z, 11E-octadecanodienoic acid (13-oxo ODE), T0070907, 5-oxo ETE, 15-oxo ETE, 15-oxo EDE, and the like, but are not limited thereto. Non-covalently bound ligands are MCC-555, 9 (S) -hydroxyoctadecadienoic acid (9 (S) -HODE), o-lysophosphaditinic acid (o-LPA), 13 (S) -hydroxyoctadedeca Preference is given to dienoic acid (13 (S) -HODE), eicosapentaenoic acid (EPA), dihomo-g-linolenic acid (DGLA), arachidonic acid. This is because such a ligand has been shown to be a ligand that does not covalently bind to a nuclear receptor, but is not limited thereto, and those skilled in the art can use various ligands based on this ligand. It will be appreciated that ligands can be made that do not covalently bind to the receptor.

  In the method for measuring the binding ability of the present invention, the target biomolecule is typically a nuclear receptor, but is not limited thereto. Since maleimide was found in the present invention to be useful for measuring covalent bonds, the present invention is useful for any receptor or protein in which covalent bonds etc. play an important role in ligand binding. It is understood that there is.

(Agonist identification method)
In accordance with the present invention, agonists that bind to a given nuclear receptor can be identified by the following steps:
A) An active site pocket of an active nuclear receptor by applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the nuclear receptor (ie, active nuclear receptor) bound to a known agonist or antagonist And B) electronically screening the spatial coordinates of the set of candidate compounds against the spatial coordinates of the active site pocket of the active nuclear receptor to obtain an active nuclear receptor Identifying candidate binding compound species that can bind.

  In step (B) of the above method, the presence or absence of a covalent bond and / or hydrogen bond (particularly a covalent bond with a specific cysteine and / or a hydrogen with arginine or tyrosine as described elsewhere herein) It is understood that the presence or absence of binding) can be considered.

  In addition to Step B above, the present invention further includes C) wherein the candidate binding compound species identified in Step (B) is a specific residue (PPARγ or a ligand-binding fragment thereof) of the activated nuclear receptor. Identifying the candidate binding compound species as a candidate agonist compound species if it has a first moiety that can interact with the tyrosine residue at position 473 (if present). Such identification can also be performed based on the method of the present invention.

  In order to carry out the above method, it is not always necessary to use the full length of the nuclear receptor, and a fragment of the nuclear receptor that binds to an agonist and / or a natural ligand may be used. .

(Identification system)
In one aspect, the present invention provides a system for identifying compounds that modulate nuclear receptors. The system includes: A) a means for determining whether a candidate compound is covalently bound to a cysteine in the nuclear receptor; and B) a compound that modulates the nuclear receptor for a candidate compound determined to be covalently bound. Means for calculating that. Examples of the covalent bond determination means used in this system include, but are not limited to, means for realizing any method described in the above method, for example, a mass spectrometer, SDS-PAGE, and the like. As a means of calculating a candidate compound determined to be covalently bound as a compound that modulates a nuclear receptor, a program that executes a procedure for deriving that a compound has a modulatory activity from data on covalent bonds is executed. Any means can be used, and examples of such means include a computer on which a program is executed.

(Measurement kit)
In another aspect, the present invention provides a kit for measuring a covalent bond between a compound and the nuclear receptor, which is used to identify a compound that modulates the nuclear receptor. The kit includes instructions that describe A) a ligand known to be irreversibly covalently bound to the nuclear receptor; and B) a procedure for measuring binding of a ligand that is not covalently bound. Here, it is understood that a compound as described above can be used as the ligand. Here, a chart or a correspondence table for deriving the state of ligand binding from the data on the covalent bond can be written in the instruction sheet. Examples of such correspondence tables include the following.

(System program)
In another aspect of the present invention, a program for executing the method of the present invention and a storage medium including such a program are also provided.

  In a further aspect of the present invention, there is provided an apparatus that can be used to perform the method of the present invention and an apparatus comprising the program of the present invention.

  In another aspect, the present invention provides a program for causing a computer to execute a method for identifying a compound that modulates a nuclear receptor. The method implemented by this program is: A) providing the conformational data of the candidate compound; and B) determining whether the candidate compound is covalently bound to a cysteine in the nuclear receptor. Identifying a candidate compound determined to be covalently bound as a compound that modulates a nuclear receptor. Here, it is understood that the data on the three-dimensional structure and the data on the binding between cysteine and the ligand can be obtained using any technique as described in the present specification based on the description in the present specification. The

  The present invention also provides a recording medium for storing such a program. As such a recording medium, any recording medium may be used as long as it can be connected so that the computer can execute the program. Such a recording medium may be, for example, a hard disk, MO, CD-R, CD-RW, CD-ROM, DVD-RAM, DVD-R, DVD-RW, DVD + RW, DVD-ROM, or memory card.

(3D structure prediction of complex of nuclear receptor and ligand)
When the three-dimensional structure of the complex of the nuclear receptor and the ligand is unknown, the three-dimensional structure can be predicted by the following method.

  As the three-dimensional structure prediction of the complex, molecular dynamics simulation, complex prediction using a docking tool, and complex prediction using Monte Carlo, molecular mechanics, or molecular dynamics simulation can be used. Examples of the docking tool include rigid body docking, semi-flexible docking, and flexible docking using a genetic algorithm and a geometric hashing algorithm. Among the multiple complex structures predicted by this docking tool, a structure having a close distance to the cysteine residue is extracted. This 3D structure prediction may include complex structures predicted by Monte Carlo, molecular mechanics, and ab initio structure prediction using molecular dynamics (non-empirical structure prediction independent of sequence and 3D structure database). .

  In addition, this three-dimensional structure prediction is based on a three-dimensional structure calculated for a complex of a main chain heavy atom of a known three-dimensional structure of the nuclear receptor and ligand complex, and the complex of the nuclear receptor and the agonist. Superposition with heavy atoms of the main chain of the structure, a complex structure with stable energy is obtained from the superposed structure. Here, when the superposed structure is a composite structure having stable energy, this is also included. Optimize structures with Monte Carlo, molecular mechanics, and molecular dynamics methods.

  For the three-dimensional structure of the agonist, canonical molecular dynamics simulation and energy minimization calculation using molecular mechanics. Monte Carlo simulations and molecular dynamics simulations including canonical, microcanonical, grand canonical and extended ensemble methods can be used.

  The simulation of the bond with the hydrogen atom in the canonical molecular dynamics simulation can be performed by applying a shake constraint or a RATTLE method.

  Handling of electrostatic interactions in Monte Carlo, molecular mechanics, and molecular dynamics methods can include no approximation, cutoff method, and multipole approximation (cell multipole).

  Canonical molecular dynamics simulation includes PRESTO program, AMBER program (UCF), CHARMM program (Harvar University), GROMOS program (BIOMOS) Discover program (Accelrys Inc), SYBYL program (Tripos Inc.), MASP For example, but not limited to.

  The agonist potential energy calculation in canonical molecular dynamics simulation includes AMBER general force field for the organic molecular parameters (parm94, parm96, parm99), OPLS, Charmm, cff, cvff and Tripos, but not limited to these.

  The charge parameter of the agonist in the canonical molecular dynamics simulation is performed using the molecular orbital calculation software Gaussian 98 to perform the structure optimization calculation of the second agonist, and to calculate the electrostatic potential of the optimized structure of the second agonist. It is determined by fitting an agonist with a RESP program. In addition to Gaussian (Gaussian Inc), GAMESS (Iowa State University), MOPAC (Fujitsu Ltd.), and the like can be used.

  The three-dimensional structure of the complex was obtained by extracting a structure compatible with the agonist binding site of the nuclear receptor from the trajectory of molecular dynamics simulation, or obtained by performing energy minimization calculation with molecular mechanics. This is done using an energy minimal structure as the composite structure.

  The three-dimensional structure prediction of the complex is performed by introducing the three-dimensional structure of the agonist into the three-dimensional structure of the nuclear receptor. If necessary, in the prediction of the three-dimensional structure of the complex, after introducing the three-dimensional structure of the agonist into the three-dimensional structure of the nuclear receptor, molecular mechanics calculation of the structural distortion of the non-covalent complex between the nuclear receptor and the agonist To eliminate.

  Eliminating structural distortion of the non-covalent complex: (i) selecting a structure in which the agonist is introduced into the nuclear receptor as an initial structure; (ii) position-constraining a molecule derived from the nuclear receptor Constraints on the initial structure by the method, freeing the atoms of the agonist to eliminate structural distortion of the entire complex by molecular mechanics; and (iii) releasing all atoms to free the entire structural distortion , Done by.

  The molecular mechanics calculation can be performed using a conjugate gradient method with a dielectric constant of 1 in a vacuum. Alternatively, in addition to the vacuum dielectric constant 1, a method that explicitly considers the solvent, a method that approximates the solvent as a continuum, and a GB-SA method can also be used. In addition to the conjugate gradient method, the steepest descent method can be used as the energy minimization algorithm of the molecular mechanics method. The calculation of electrostatic interaction in the molecular mechanics calculation may be performed by capping the N-terminal of the nuclear receptor peptide with an acetyl group and the C-terminal with an N-methyl group. The calculation of the electrostatic interaction in the molecular mechanics calculation may be performed with the aspartic acid residue, glutamic acid residue, arginine residue, lysine residue, and histidine residue of the nuclear receptor in a charged state. The calculation of the electrostatic interaction in the molecular mechanics calculation may be performed with the carboxyl group of the agonist as a charged state.

  The molecular mechanics calculation includes PRESTO program, AMBER program (UCF), CHARMM program (Harvar University), GROMOS program (BIOMOS) Discover program (Accelrys Inc), SYBYL program (Tripos Inc), and MASPHYC program (Fujitsu). However, it is not limited to these. Molecular mechanics calculations include, but are not limited to, AMBER general force field for the organic molecular parameters (parm94, parm96, parm99), OPLS, Charmm, cff, cvff and Tripos.

  In the prediction of the three-dimensional structure of the complex, even if a canonical molecular dynamics simulation is carried out with the structure after the structural distortion of the non-covalent complex of the nuclear receptor and the agonist is eliminated by molecular mechanics calculation as the initial structure. Good.

  The canonical molecular dynamics simulation is performed in a vacuum, a dielectric constant of 1, and a temperature of 300K. Alternatively, in addition to using the vacuum dielectric constant 1, a method that explicitly considers the solvent, a method that approximates the solvent by a continuum, and a GB-SA method may be used. As an algorithm for minimizing energy in the molecular mechanics method, the steepest descent method may be used in addition to the conjugate gradient method.

  The canonical molecular dynamics simulation can be performed by constraining the main chain of the nuclear receptor to the initial structure by the position constraint method. Further, in the canonical molecular dynamics simulation, the simulation of the bond with the hydrogen atom may be performed by applying a shake constraint.

(Identification of agonist)
In accordance with the present invention, for example, but not limited to, agonists that bind to a given nuclear receptor can be identified as follows:
A) Applying a three-dimensional molecular modeling algorithm to the atomic coordinates of a nuclear receptor bound to a known agonist and / or antagonist, that is, the atomic coordinates of an active nuclear receptor, Determining the spatial coordinates of the active site pocket of
B) Electronically screening the spatial coordinates of the set of candidate compounds against the spatial coordinates of the active site pocket of the active nuclear receptor to bind to the active nuclear receptor or a ligand-binding fragment thereof. Identifying a candidate binding compound species to be obtained; and C) identifying a compound species having an agonistic action as a candidate agonist compound species from among the candidate binding compound species identified in step (B) above.

  The atomic coordinates of the active nuclear receptor can be determined from the three-dimensional structure of a complex of a known agonist and nuclear receptor, or a complex of a natural ligand and nuclear receptor. When the three-dimensional structure of the complex is unknown, the three-dimensional structure of the complex can be predicted according to the above “prediction of the three-dimensional structure of the complex of the nuclear receptor and the ligand”.

  In the identification of an agonist, whether or not a candidate compound can bind to a nuclear receptor is determined based on whether or not the three-dimensional structure of the active site pocket of the nuclear receptor is a structure that binds the candidate compound. decide.

  In the above method, it is not always necessary to use the full length of the nuclear receptor. Instead of the full length of the nuclear receptor, a fragment of a nuclear receptor that can bind to a known agonist and / or a natural ligand may be used. it can.

  When the predetermined nuclear receptor is PPARγ, instead of the step (C), as the step (C ′), the candidate binding compound species identified in the step (B) is an active PPARγ or A step of identifying candidate agonist compound species from candidate binding compound species can be used by using as an index whether or not the ligand binding fragment has a first portion capable of interacting with the tyrosine residue at position 473. Alternatively, in place of the step (C), as a step (C ″), the candidate binding compound species identified in the step (B) interacts with a tyrosine residue at position 473 of the active PPARγ or a ligand-binding fragment thereof. Candidate binding compounds, using as an index whether or not they have a first part that can act and a second part that can be covalently bound to a cysteine residue at position 285 of active PPARγ or a ligand-binding fragment thereof A step of identifying a candidate agonist compound species from the species can be used. For other nuclear receptors, the same identification can be performed by using a correspondence table such as Table 4.

Alternative methods for identifying agonists include, but are not limited to, for example, comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of an active nuclear receptor and The process can be used to identify compounds that can bind to active nuclear receptors:
A) applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the activated nuclear receptor to obtain a three-dimensional molecular model;
B) Inputting coordinate data of the three-dimensional molecular model into a data structure and searching for a distance between atoms of the active nuclear receptor;
C) Comparing the distance between the heteroatom that forms a hydrogen bond in the candidate compound and the heteroatom that forms the active site pocket in the three-dimensional molecular model, the optimal hydrogen bond between the two structures Identifying a candidate binding compound species that theoretically forms a stable complex with the active site pocket of the active nuclear receptor three-dimensional molecular model; and D) candidates identified in step (C) above Identifying a compound species having an agonistic action from among the binding compound species as a candidate agonist compound species.

  The atomic coordinates of the active nuclear receptor can be determined from the three-dimensional structure of a complex of a known agonist and nuclear receptor, or a complex of a natural ligand and nuclear receptor. When the three-dimensional structure of the complex is unknown, the three-dimensional structure of the complex can be predicted according to the above “prediction of the three-dimensional structure of the complex of the nuclear receptor and the ligand”.

  In the above method, it is not always necessary to use the full length of the nuclear receptor. Instead of the full length of the nuclear receptor, a fragment of a nuclear receptor that can bind to a known agonist and / or a natural ligand may be used. it can.

  When the predetermined nuclear receptor is PPARγ, instead of the step (D), as the step (D ′), the candidate binding compound species identified in the step (C) is an active PPARγ or A step of identifying candidate agonist compound species from candidate binding compound species can be used by using as an index whether or not the ligand binding fragment has a first portion capable of interacting with the tyrosine residue at position 473. Alternatively, as a step (D ″) instead of the step (D), the candidate binding compound species identified in the step (C) interacts with a tyrosine residue at position 473 of the active PPARγ or a ligand-binding fragment thereof. Candidate binding compounds, using as an index whether or not they have a first part that can act and a second part that can be covalently bound to a cysteine residue at position 285 of active PPARγ or a ligand-binding fragment thereof A step of identifying a candidate agonist compound species from the species can be used.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited thereto.

(Example 1: Expression and purification of recombinant nuclear receptor ligand binding domain (LBD) in E. coli)
Using a recombinant nuclear receptor ligand binding domain (LBD) expressed and purified in E. coli, the increase in molecular weight accompanying irreversible binding of the ligand was measured using a time-of-flight mass spectrometer (TOF-MS) ( FIG. 4).

  Human PPARγLBD (aa. 195-477) was cloned into the NdeI / BamHI site of pET28b manufactured by Novagen, and the nucleotide sequence was confirmed by sequencing. Escherichia coli BL21 (DE3) was transformed and cultured with 1 L of LB at 37 ° C. until OD600 reached 0.8, then transferred to 18 ° C., and 1 mM IPTG was added and cultured for 18 hours. E. coli was collected by centrifugation, suspended in 20 mM Hepes pH 7.4, 200 mM NaCl, 25 mM imidazole, and stored at −20 ° C.

  The cells were sonicated, insoluble proteins were removed by centrifugation, and PPARγLBD was purified by affinity chromatography using a nickel column. At the same time as purification by Superdex 200 gel filtration chromatography, the buffer was exchanged to 20 mM Tris pH 7.4, 150 mM NaCl. The obtained PPARγLBD was concentrated by ultrafiltration.

The TOF-MS measurement was performed as follows. PPARγLBD and the ligand were mixed in a buffer of 20 mM Tris pH 7.4, 150 mM NaCl and incubated at room temperature for 30 minutes to form a complex. The composite solution 1 was mixed with a 4-fold amount of sinopic acid solution (33% acetonitrile), dried, and then subjected to TOF-MS measurement. The mass spectrometry spectrum was measured using Voyager MALDI-TOF-MS. Various parameters for measurement are as follows.
mode: linear
accelerating voltage: 20000
grid voltage: 90.400%
guid wire voltage: 0.200%
delay: 200 on
laser: 3000
scan averaged: 188
pressure: 3.98e-7
PPARγLBD mixed with the same concentration of DMSO solution in which the ligand was dissolved was used as a control.

(Quantitative measurement of binding using fluorescent maleimide reagent)
Measurement of ligand binding activity using recombinant PPARγLBD. The irreversible binding of the agonist to the cysteine residue present at the ligand binding site was quantitatively measured using a fluorescent maleimide reagent (FIG. 5).

  After mixing 0.1 μM PPARγLBD with various concentrations of ligand and incubating at room temperature for 30 minutes, SDS was added at 0.5%, rhodamine maleimide (Molecular Probe) at 200 μM, and TCEP at 1 mM, and room temperature was added. And incubated for 30 minutes. After performing SDS-PAGE, the amount of PPARγLBD labeled with rhodamine maleimide was measured with a fluorescence imager (FMBIO II manufactured by Hitachi).

By using the above method was measured for various ligands, among the compounds shown in FIG. 6, in particular, _15d-PGJ 2, CAY, 9-oxo ODE and 13-oxo ODE was found to be covalently bound .

(Example 2: Practice of theoretical calculation protocol)
(1: Prediction of three-dimensional structure of PPARγ-15d-PGJ ₂ noncovalent complex)
Since the crystal structure of PPARγ and its natural ligand 15d-PGJ ₂ complex has not been reported so far, the PPARγ-agonist complex 4 structure (Protein Data Bank ID code: 1fm6 [Chen et al. 2000] reported previously). , 1i7i [Clonet et al. 2001], 1 knu [Sauerberg et al. 2002], 4prg [Oberfield et al. 1999]). In order to analyze the binding mode of agonists, they were overlaid with heavy atoms in the main chain of PPARγ (FIG. 7a). The structure of the agonist complex is shown in FIGS. In region 1 of FIG. 7a, the nitrogen of the amide group and oxygen of the carboxyl group of the agonist were present, and in the region 2, hydrocarbons and benzene rings were present. From this result, it was predicted that the carboxyl group of 15d-PGJ ₂ was present in region 1 of FIG.

A plurality of 15d-PGJ ₂ structures were prepared from a canonical molecular dynamics simulation in a temperature of 500 K and in a vacuum. The temperature was raised from 0K to 500K in 100,000 steps, and then sampling at 500K for 5 million steps (5 ns). In the sampling calculation, the structure was saved to a file every 1000 steps. The time increment was 1 fs and the dielectric constant was 80. SHAKE constraint on the bond with hydrogen atom [Ryckerert et al. 1977] was applied. All 1-4, 1-5 pairs of electrostatic interactions were calculated without approximation. The carboxyl group of 15d-PGJ ₂ was charged. For calculating molecular dynamics, PRESTO ver. 3 (Biomolecular Engineering Research Institute [Morikami et al. 1992]) program was used. Of the potential energy calculations 15d-PGJ _2, using the AMBER general force field for the organic molecule parameters [http://amber.scripps.edu/]. However, since the parameter of the charge of 15d-PGJ ₂ has not been reported so far, using the molecular orbit calculation software Gaussian 98 (Gaussian Inc. [Frisch et al. 1998]), Hartley-Fock / 6- 6 The structure optimization calculation of 15d-PGJ ₂ is executed with 31G *, and the electrostatic potential of the optimized structure is newly fitted by RESP (AMBER group [Bayly et al. 1993, Cornell et al. 1993]) program. The charge was determined.

  One structure that fits into the PPARγ ligand binding pocket was extracted from the trajectory of the molecular dynamics simulation. Molecular drawing software Insight II (Accelry Inc.) was used for the structure extraction work.

15d-PGJ ₂ was introduced into PPARγ. 1fm6 [Chen et al. 2000] was used as a template for the PPARγ-15d-PGJ ₂ complex. 1fm6 is a heterodimeric complex of a retinoid X receptor and PPARγ, and this structure includes a coactivator peptide bound to PPARγ and an agonist BRL (hSRC1 = ERHKILHRLLQEGSPS (SEQ ID NO: 47)). Here, the D chain of PPARγ, the BRL bound thereto and the coactivator peptide were excised and used for introduction of 15d-PGJ ₂ into PPARγ. Based on the above prediction of the arrangement of 15d-PGJ ₂ in PPARγ, the amide group or carbonyl group part of BRL (FIG. 7b, region 1) has a carboxyl group of 15d-PGJ ₂ and the skeleton part of BRL (FIG. 7b, region). 15d-PGJ ₂ extracted above was introduced so that the backbone of the chain having a carboxyl group of 15d-PGJ ₂ was fitted to ₂ ) (FIG. 8). For the introduction work, molecular drawing software Insight II (Accelry Inc.) was used.

The structural distortion of the PPARγ-15d-PGJ ₂ non-covalent complex was eliminated by a two-stage molecular mechanics calculation. For the initial structure, a complex into which 15d-PGJ ₂ was introduced was used. In the first stage, other than 15d-PGJ ₂ was restrained with respect to the initial structure by the position restraint method, and in the second stage of 15d-PGJ ₂ , all atoms were freed to eliminate the overall structural distortion. Molecular mechanics calculations were performed in vacuum using a conjugate gradient method with a dielectric constant of 1. The electrostatic interaction is performed by the cell multipole method [Ding et al. 1992]. The N-terminus of PPARγ and coactivator peptide was capped with an acetyl group, and the C-terminus of the coactivator peptide was capped with an N-methyl group. Aspartic acid PPARy, was glutamic acid, arginine, lysine, histidine, carboxyl group of 15d-PGJ ₂ is a charged state. For molecular mechanics calculations, PRESTO ver. 3 (Biomolecular Engineering Research Institute [Morikami et al. 1992]) program was used. For calculating the potential energy of PPARγ and coactivator peptides, AMBER param 96 parameter [Kollman et al. 1997], the potential energy calculations 15d-PGJ _2, and using the above AMBER general force field for the organic molecule Parameters [Http://Amber.Scripps.Edu/] and charge.

A canonical molecular dynamics simulation was performed on the PPARγ-15d-PGJ ₂ composite at a vacuum, a dielectric constant of 1, and a temperature of 300K. The structure obtained from molecular mechanics calculation was used as the initial structure. The main chains of PPARγ and coactivator peptide were constrained to the initial structure by the position constraint method. The temperature was raised from 0K to 300K in 100,000 steps, and then 2 million steps (2 ns) were sampled at 300K. The time increment was 1 fs. In the sampling calculation, the structure was saved every 1000 steps. A SHAKE constraint [Rykkaert 1977] was applied to the bond with the hydrogen atom. The handling of electrostatic interaction, calculation system, potential energy parameters, and calculation program were the same as the molecular mechanics calculation.

The trajectory of the molecular dynamics simulation of the PPARγ-15d-PGJ ₂ complex was analyzed. Cys285 of PPARγ is positioned in the vicinity of the 5-membered ring of 15d-PGJ _2. 15d-PGJ ₂ is known to be covalently bonded to a molecule having a thiol group such as cysteine by a Michael addition reaction. Site of a possible reaction of 15d-PGJ ₂ is two ways. Were plotted distances d1, d2 of Cys285 and 15d-PGJ ₂ shown in FIG. 9b, respectively ~3.5, ~5.5Å next (Fig. 9a), Cys285 and 15d-PGJ ₂ is in a position to covalently bond It was predicted that there would be. The structure of the noncovalent bond is shown in FIG.

(2: Model construction of PPARγ-15d-PGJ ₂ covalent complex)
Cys285 and 15d-PGJ ₂ of PPARγ from binding experiments were shown to covalently bond. Considering stereoisomers, PPARγ Cys285 and 15d-PGJ ₂ can take four different modes of binding (FIG. 10). A binding experiment of compounds CAY and PPARγ in which the double bond of the 5-membered ring portion of 15d-PGJ ₂ was replaced with a single bond revealed that CAY was covalently bound to Cys285 of PPARγ and 15d-PGJ ₂ ( FIG. 6). Thus, Cys285 of PPARγ is expected to take a binding mode of 15d-PGJ ₂ and III and IV. From the molecular dynamics simulation of one non-covalent complex, Cys285 was in IV position relative to PPARγ in the protein. Therefore, a model of a PPARγ-15d-PGJ ₂ covalent complex having an IV binding mode was constructed, and PPARγ residues important for interaction with the ligand were identified.

Starting from the complex structure of 15d-PGJ ₂ introduced into PPARγ in 1 above (the structure before the molecular mechanics calculation), the bond order of Cys285, 15d-PGJ ₂ of this structure was modified to the covalent bond state IV. . For the correction, molecular drawing software Insight II (Accelry Inc.) was used. Thereafter, it defined Cys285, and this and the 15d-PGJ ₂ covalently linked as a single residue, referred to as Cyp285.

Molecular mechanics calculations were performed on the complex structure with the bond order corrected using the same protocol as 1 to eliminate structural distortion. However, in the first-stage molecular mechanics calculation, atoms on the main chain side from the sulfur atom of Cyp285 and all other protein and peptide residues were constrained by the position constraint method. Further, the carboxyl group of Cyp285 was charged. In calculating the potential energy of Cyp285, the AMBER general force field for the organic molecule parameter [http://amber.scripps.edu/] was used. The parameters of the charge of Cyp285 calculated newly at 15d-PGJ ₂ and the same method.

A canonical molecular dynamics simulation was performed on the PPARγ-15d-PGJ ₂ covalently bonded complex obtained by molecular mechanics calculation at a vacuum, a dielectric constant of 1, and a temperature of 300K. The main chains of PPARγ and coactivator peptide were constrained to the initial structure by the position constraint method. The temperature was raised from 0K to 300K by 100,000 steps, then 300K was equilibrated at 500,000 steps (500 ps), and 300K was sampled at 500,000 steps. The time increment was 1 fs. The rest of the protocol was the same as the molecular dynamics simulation of 1 PPARγ-15d-PGJ ₂ noncovalent complex.

A model structure was constructed from the trajectory of the molecular dynamics simulation of the PPARγ-15d-PGJ ₂ noncovalent complex (FIG. 11). The average structure of all the sampled structures was obtained, and the structure closest to the average structure was taken as the model structure. The root-mean-square deviation of all the PPARγ atoms in the model structure and the average structure was 0.36Å. Moreover, the root mean square dispersion of the PPARγ total heavy atoms of the model structure and the template structure (1fm6) was 1.48%.

In the model structure of the PPARγ-15d-PGJ ₂ non-covalent complex, it was revealed that the carboxyl group of the 15d-PGJ ₂ portion of Cyp285 and the hydroxyl group of the side chain of Tyr473 were hydrogen-bonded (FIG. 12). Tyr473 is the residue at helix 12. In the nuclear receptor, it is known that the position of helix 12 is different between the active type and the inactive type. The structure used for the molecular dynamics simulation of the PPARγ-15d-PGJ ₂ non-covalent complex is an active structure. From the above, it is predicted that the hydrogen bond between Tyr473 and Cyp285 is important for keeping the structure of helix 12 in an active form. Mutation experiment of Tyr473Phe was conducted, and the result was that the activity of PPARγ observed in the wild strain was lost in the mutant. The experimental results suggest that there is some interaction between the hydroxyl group and 15d-PGJ ₂ of Tyr473, to support the calculation result.

(Example 3: Quantitative measurement of binding ability of covalently bound ligand)
Various concentrations of covalently bound ligand 5 μM were added to PPARγLBD to 0.5 μM and incubated at room temperature for 15 minutes, and then 0.5% SDS, 200 μM rhodamine maleimide, and 1 mM TCEP. In addition, it was incubated at room temperature for 30 minutes. After performing SDS-PAGE, the amount of PPARγLBD labeled with rhodamine maleimide was measured with a fluorescence imager. The results are shown in FIG. 13A.

  The binding capacity was calculated as follows:

It is defined as
The reaction rate equation for

It is expressed. Solving this differential equation,

According to this equation, fitting is performed by a non-linear least square method using k as a variable to obtain an optimum k.
(Data processing)
Using Microsoft Excel, data was fitted according to the kinetics of irreversible binding, and various reaction constants were obtained. The results are shown in FIG. 13A (right).

Various parameters of the nonlinear least squares method are as follows.
Limit time 10000 seconds Repeat 10000
Accuracy 0.000001
Tolerance 2%
Convergence 0.00001
Approximation formula First-order differential coefficient Forward search method Quasi-Newton method.

(Example 4: Quantitative measurement of binding ability of ligand not covalently bound)
Various concentrations of non-covalent ligands were mixed with 5 μM 15d-PGJ ₂ and then PPARγLBD was added to 0.5 μM and incubated at room temperature for 15 minutes, then SDS was 0.5% and rhodamine maleimide was 200 μM. , And TCEP were added to 1 mM and incubated at room temperature for 30 minutes. After performing SDS-PAGE, the amount of PPARγLBD labeled with rhodamine maleimide was measured with a fluorescence imager.

  The binding capacity was calculated as follows:

It is defined as

So

Organizing this for [af]
And expressed by a quadratic equation for [af]. From the quadratic equation,

here,

Considering that NR in is occupied by competitors,

So

Since [af] includes x, it is difficult to obtain an analytical solution. Therefore, a solution of the difference equation at time t is generated using Ka as a variable, and compared with actual data, an optimum Ka is obtained by the nonlinear least square method.

(Data processing)
Data fitting according to the kinetics of irreversible binding was performed using Microsoft Excel, and various reaction constants were obtained. The results are shown in FIG. 13B (right).
Various parameters of the nonlinear least squares method are as follows.
Time limit 1000 seconds repetition 10000
Accuracy 0.0000001
Tolerance 5%
Convergence 0.0000000001
Approximation formula First-order differential coefficient Forward search method Quasi-Newton method.

(Example 5: Measurement of ligand transcriptional activity using luciferase assay in cultured cells)
The transcriptional activity of the ligand was measured using a luciferase assay in cultured cells (FIG. 14).

COS-7 cells were cultured in culture medium DMEM / 10% FCS. The day before transfection, cells were seeded in a 24-well plate at 5 × 10 ⁴ cells / well, and the following day, UAS-tk-Luc, pEYFP-C1, and pCMX-gal-PPARγ plasmids were transfected, and 6 hours later Medium was changed and various ligands were administered. Cells were collected 24 hours after transfection, and the enzyme activity of the expressed luciferase was measured by chemiluminescence using luciferin as a substrate. The fluorescence of YFP was measured and used as transfection efficiency.

(Example 6: Introduction of point mutation into PPARγLBD)
PCMX-gal-PPARγ in which PPARγLBD has been cloned was subjected to PCR using sense and antisense oligonucleotides mutated, the template plasmid was digested with DpnI, transformed into E. coli DH5α, and plated on an LB plate. . The plasmid was recovered from the colony, and mutation introduction was confirmed by sequencing.

Replacing cysteine 285 with alanine, valine, and serine significantly reduced the agonist activity of 15d-PGJ ₂ and 13-oxoODE, which was concluded to be covalently linked to cysteine (FIG. 15). This result indicates that binding to cysteine is important for agonist activity by 15d-PGJ ₂ and 13-oxoODE.

  In addition, when tyrosine 473 was substituted with phenylalanine, none of the compounds showed agonistic activity. This indicates that tyrosine 473 is essential for the activation of PPARγ regardless of the presence or absence of a covalent bond (FIG. 16).

Example 7: Assay of actual compounds
Of the compounds represented by R ₁ —CH═CH— (C═O) —R ₂ —R ₃

R ₁ : C13 (—CH═CH—CH ₂ —CH═CH—CH ₂ —CH═CH—CH ₂ —CH ₂ —CH ₂ —CH ₂ —CH ₃ )
_{R 2: C4 (-CH 2 -CH} 2 -CH 2 -C-)
R ₃ : hydroxyl group (—OH), carbonyl group (C═O)

It was clarified in an activity measurement system using luciferase in a cultured cell as a reporter gene that 5-oxoETE constituted by is an agonist for PPARγ.

  The above compound was purchased commercially (obtained from Caymanchem).

  The assay was performed as described in the examples above.

  As a result of the assay, the activities of analog 5-HETE and arachidonic acid having no α, β unsaturated ketone were low (FIG. 19).

  To date, there has been no report that 5-oxoETE is a ligand of PPARγ, and it can be said that the proposal of the present inventors has led to the identification of an unknown ligand.

Example 8: Another compound example
Among the compounds represented by R ₁ — (C═O) —CH═CH—R ₂ —R ₃ , R1: C ₄ (—CH ₂ —CH ₂ —CH ₂ —CH ₃ )
_{R2: C 12 (-CH = CH} -CH 2 -CH = CH-CH 2 -CH = CH-CH 2 -CH 2 -CH 2 -C-)
R3: hydroxyl group (—OH), carbonyl group (C═O)
The activity measurement system using luciferase in a cultured cell as a reporter gene revealed that 15-oxoETE composed of Here, this compound was purchased from Caymanchem. The assay was performed as described in the examples above. As a result, analogs 15-HETE and arachidonic acid having no α, β unsaturated ketone had low activity (FIG. 20).

  So far, there is no report that 15-oxoETE is a ligand of PPARγ, and it can be said that the proposal of the present inventors has led to the identification of an unknown ligand.

Example 9: Further compound examples
Among the compounds represented by R ₁ — (C═O) —CH═CH—R ₂ —R ₃ , R1: C4 (—CH ₂ —CH ₂ —CH ₂ —CH ₃ )
R2: C12 (—CH═CH—CH ₂ —CH ₂ —CH ₂ —CH ₂ —CH ₂ —CH ₂ —CH ₂ —CH ₂ —CH ₂ —C—)
R3: hydroxyl group (—OH), carbonyl group (C═O)
It was clarified in an activity measurement system using luciferase in a cultured cell as a reporter gene that 15-oxoEDE constituted by is an agonist for PPARγ.

  The above compound was purchased commercially (obtained from Caymanchem).

  The assay was performed as described in the examples above.

  As a result, analogs 15-HEDE and arachidonic acid having no α, β unsaturated ketone had low activity (FIG. 20).

  Similarly, for indomethacin, when a compound obtained from Sigma was tested, the agonist activity was similarly confirmed.

  To date, there is no report that 15-oxoEDE is a ligand of PPARγ, and it can be said that our proposal has led to the identification of an unknown ligand.

(References)
Chen, S., et al., (2000), J. et al. Biol. Chem. 275, 3733-3736.
Cronet, P., Peterson, JFW, Folmer, R., Blomberg, N., Sjoblom, K., Karlsson, U., Lindstedt, E.-L. & Bamberg, K.C. (2001), Structure 9, 699-706.
Sauerberg, P., et al., (2002) J. Am. Med. Chem. 45, 789-804
Oberfield, JL, et al., (1999) Proc. Natl. Acad. Sci. USA 96 6102-6.
Ryckaert, J.-P., Ciccotti, G. & Berendsen, HJC. (1977) Numerical interaction of the Cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes. J. Comput. Phys. 23, 327-341.
Morikami, K., et al., (1992) Comput. Chem. 16, 243-248.
Frisch, MJ, et al., (1998) Gaussian 98, Revision A.11.3. Gaussian, Inc., Pittsburgh, PA.
Bayly, CI, et al., (1993), J. et al. Phys. Chem. 97, 10269-10280.
Cornell, WD, et al., (1993) J. Am. Am. Chem. Soc. 115, 9620-9631.
Ding, H.-Q., et al., (1992) J. et al. Chem. Phys. 97, 4309-4315.
Kollman, P. et al., (1997) Computer simulations of biomolecular systems (eds. WF. Van Gunsteren, PK. Weiner, and AJ. Wilkinson), vol. 3, pp. 83-96. KLUWER / ESCOM, TheNetherlands.
As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, a method for identifying a compound that modulates a nuclear receptor is provided, and a modulator of the nuclear receptor identified thereby is also provided. It will be appreciated that such regulation of nuclear receptors is useful for the regulation of basic biological functions and is therefore useful in various fields. It is understood that such fields are useful in fields such as agriculture, medicine, pharmacy (especially the pharmaceutical industry), biotechnology, and related industries.

  Furthermore, in accordance with the present invention, a method is provided that, when given an agonist for a nuclear receptor, increases the affinity of the agonist for the nuclear receptor without substantially compromising the activity of the agonist. Also provided are methods of designing agonists with increased affinity using such methods. Such methods are used to provide agonists with increased affinity. A program used in such a method, a storage medium containing such a program, and a computer containing such a program are also provided.

  Also provided by the present invention are agents for treating diseases associated with nuclear receptors, particularly PPARγ related diseases.

FIG. 1 is a schematic diagram of the structure of PPARγ. FIG. 2-1 is an alignment of ligand binding sites of PPARγ and other nuclear receptors. FIG. 2-2 shows the alignment of ligand binding sites of PPARγ and other nuclear receptors. FIG. 2-3 is a continuation of alignment of ligand binding sites of PPARγ and other nuclear receptors. FIG. 3 is a diagram showing representative PPARγ agonists. FIG. 4 is a diagram showing the results of TOF-MS analysis of ligand binding. FIG. 5 is a diagram showing the results of quantification of the covalent bond of a ligand with a fluorescent maleimide reagent. FIG. 6 is a diagram showing the results of searching for a basic skeleton common to covalently bound ligands. FIG. 7 is a diagram showing the result of superposition of heavy atoms in the main chain of PPARγ. The structure of the agonist complex is shown in FIGS. FIG. 8 is a diagram showing the results of PPARγ with 15d-PGJ ₂ introduced. Figure 9 is a result showing the calculation result of the distance between Cys285 and 15d-PGJ ₂ of PPARy. Figure 10 is a diagram showing the binding mode can be between Cys285 and 15d-PGJ ₂ of PPARy. FIG. 11 is a diagram showing the structure of the PPARγ-15d-PGJ2 covalent complex obtained as a result of the simulation of Example 2. FIG. 12 is a diagram schematically showing hydrogen bonding between Tyr473-Cyp285 in the final structure of the PPARγ-15d-PGJ ₂ covalent bond complex. Hydrogen bonds are indicated by broken lines. FIG. 13A is a diagram showing the results of quantification of the binding ability of a covalently binding ligand and the results of a binding assay using a covalently binding ligand. FIG. 13B shows the results of various reaction constants obtained by fitting data according to the kinetics of irreversible binding by the least square method, as shown in Example 4. FIG. 14 is a diagram showing the results of measurement of ligand transcriptional activity by luciferase assay in cultured cells. FIG. 15 is a diagram showing the results of examining the necessity of covalent bonding using cysteine mutants. FIG. 16 is a diagram showing the results of confirmation of the function of tyrosine 473. FIG. 17 shows the final structure of the PPARγ-15d-PGJ ₂ non-covalent complex. FIG. 18 shows the final structure of the PPARγ-15d-PGJ ₂ covalent complex. FIG. 19 shows the assay results in Example 7. FIG. 20 shows the assay results in Examples 8 and 9.

SEQ ID NO: 1: ERR1 nucleic acid sequence.
SEQ ID NO: 2: amino acid sequence of ERR1.
SEQ ID NO: 3: Nucleic acid sequence of ERR2.
SEQ ID NO: 4: amino acid sequence of ERR2.
SEQ ID NO: 5: ERR3 nucleic acid sequence.
SEQ ID NO: 6: amino acid sequence of ERR3.
SEQ ID NO: 7: Nucleic acid sequence of LRH1.
SEQ ID NO: 8: amino acid sequence of LRH1.
SEQ ID NO: 9: SF1 nucleic acid sequence.
SEQ ID NO: 10: amino acid sequence of SF1.
SEQ ID NO: 11: Nucleotide sequence of RXRα.
SEQ ID NO: 12: amino acid sequence of RXRα.
SEQ ID NO 13: Nucleic acid sequence of RXRβ.
SEQ ID NO: 14: amino acid sequence of RXRβ.
SEQ ID NO: 15: Nucleic acid sequence of RXRγ.
SEQ ID NO: 16: amino acid sequence of RXRγ.
Sequence number 17: The nucleic acid sequence of HNF4 (alpha).
SEQ ID NO: 18: Amino acid sequence of HNF4α.
SEQ ID NO: 19: Nucleic acid sequence of HNF4γ.
SEQ ID NO: 20: Amino acid sequence of HNF4γ.
SEQ ID NO: 21: nucleic acid sequence of RARα.
SEQ ID NO: 22: amino acid sequence of RARα.
SEQ ID NO: 23: nucleic acid sequence of RARβ.
SEQ ID NO: 24: Amino acid sequence of RARβ.
SEQ ID NO: 25: nucleic acid sequence of RARγ1.
SEQ ID NO: 26: Amino acid sequence of RARγ1.
SEQ ID NO: 27: nucleic acid sequence of RARγ2.
SEQ ID NO: 28: Amino acid sequence of RARγ2.
SEQ ID NO: 29: nucleic acid sequence of RORα.
SEQ ID NO: 30: Amino acid sequence of RORα.
SEQ ID NO: 31: nucleic acid sequence of RORβ.
SEQ ID NO: 32: amino acid sequence of RORβ.
SEQ ID NO: 33: nucleic acid sequence of RORγ.
SEQ ID NO: 34: amino acid sequence of RORγ.
SEQ ID NO: 35: nucleic acid sequence of O43245.
SEQ ID NO: 36: amino acid sequence of O43245.
SEQ ID NO: 37: nucleic acid sequence of PPARα.
SEQ ID NO: 38: Amino acid sequence of PPARα.
SEQ ID NO: 39 nucleic acid sequence for PPARβ / δ.
SEQ ID NO: 40: amino acid sequence of PPARβ / δ.
SEQ ID NO: 41: nucleic acid sequence of PPARγ1.
SEQ ID NO: 42: amino acid sequence of PPARγ1.
SEQ ID NO: 43: nucleic acid sequence of PPARγ2.
SEQ ID NO: 44: amino acid sequence of PPARγ2.
SEQ ID NO: 45: TLX nucleic acid sequence.
SEQ ID NO: 46: Amino acid sequence of TLX.
SEQ ID NO: 47: Sequence of hSRC1

Claims (33)

A method of identifying a compound that modulates a nuclear receptor, the method comprising:
A) providing a candidate compound; and B) determining whether the candidate compound is covalently bound to cysteine in the nuclear receptor, wherein the candidate compound determined to be covalently bound is Identifying a compound that modulates an internal receptor,
Including the method. The method of claim 1, wherein the cysteine is present in a ligand binding domain. The nuclear receptors are PPARα, PPARβ / δ, PPARγ1, PPARγ2, ERR1, ERR2, ERR3, HNF4α, HNF4β, RARα, RARβ, RARγ1, RARγ2, RXRα, RXRβ, RXRγ, RORα, RORβ, ROR1F 2. The method of claim 1, wherein the method is selected from the group consisting of TLX and O4245 receptors. The method according to claim 1, wherein the nuclear receptor is PPARγ. The position of the cysteine is shown in the table below.
The method according to claim 1, which is represented by an amino acid number in SEQ ID NO: The candidate compound includes a compound having a substituent of R _A —CH═CH— (C═O) —R _B — or R _A — (C═O) —CH═CH—R _B —, wherein R _A is a monovalent substituent containing hydrogen or an optionally substituted substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group, and a thiol group, and R _B is a divalent carbonization The method according to claim 1, which is a hydrogen group. The method according to claim 1, wherein the measurement of the covalent bond is performed using a compound called fluorescently labeled maleimide. The method of claim 1, further comprising determining whether the candidate compound hydrogen bonds with an amino acid in helix 12 or helix 5 in the ligand binding domain of the nuclear receptor. The amino acid residue number in helix 12 or helix 5 is
The method of Claim 8 containing the arginine or tyrosine shown by the amino acid number in sequence number shown by these. The method according to claim 8, wherein the amino acid in helix 12 or helix 5 comprises tyrosine 473 of the amino acid sequence shown in SEQ ID NO: 42 or tyrosine 501 of the amino acid sequence shown in SEQ ID NO: 44. The method according to claim 8, wherein the hydrogen bond with the specific amino acid is measured by means of a nuclear magnetic resonance apparatus (NMR) or an X-ray diffractometer. The method according to claim 1, wherein the covalent bond with the specific amino acid is measured by means of a mass spectrometer or SDS-PAGE. The method of claim 1, wherein the modulation is inhibition. The method of claim 1, wherein the adjustment is facilitation. A compound identified by the method of claim 1, wherein the compound has the following formula:
(Chemical formula 1)
R ₁ —CH═CH— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —CH═CH—R ₂ —R ₃
Where R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 13 carbon atoms connecting the shortest distance, and R ₂ is A saturated or unsaturated hydrocarbon group which may be substituted, and R ₃ represents a substituent selected from the group consisting of hydrogen or an optionally substituted carbonyl group, hydroxyl group, amide group and thiol group; A compound in which R ₂ and R ₃ are monovalent substituents, and the maximum number of carbon atoms connecting the shortest distance is 4 to 14 for the contained carbon. In the compound,
R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 9 carbon atoms connecting the shortest distance, and R ₂ is an optionally substituted saturated or An unsaturated hydrocarbon group, and R ₃ is a monovalent hydrocarbon group containing a substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group and a thiol group, wherein R ₂ and R ₃ Is the compound of Claim 15 whose maximum value of the carbon number which connects the shortest distance is 6-12 pieces about carbon contained. A composition for modulation of a nuclear receptor comprising a specified compound according to the method of claim 1, wherein the compound has the formula:
(Chemical formula 2)
R ₁ —CH═CH— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —CH═CH—R ₂ —R ₃
Where R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 13 carbon atoms connecting the shortest distance, and R ₂ is A saturated or unsaturated hydrocarbon group which may be substituted, and R ₃ represents a substituent selected from the group consisting of hydrogen or an optionally substituted carbonyl group, hydroxyl group, amide group and thiol group; A composition in which R ₂ and R ₃ are monovalent substituents, and the maximum number of carbon atoms connecting the shortest distance is 4 to 14 with respect to the contained carbon. The structure is
R ₁ — (C═O) —CH═CH—R ₂ —R ₃ ,
In the compound,
R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 9 carbon atoms connecting the shortest distance, and R ₂ is an optionally substituted saturated or unsaturated hydrocarbon group. A saturated hydrocarbon group, R ₃ is a monovalent substituent containing hydrogen or an optionally substituted substituent selected from the group consisting of a carbonyl group, a hydroxyl group, an amide group and a thiol group; Here, for R ₂ and R ₃ , the maximum number of carbons that connect the shortest distance is 6 to 14 for the contained carbon,
The combination is
The composition of Claim 17 which has carbon number of the right column with respect to the nuclear receptor which has an amino acid sequence shown to sequence number as described in. A composition for the treatment, prevention or prognosis of a disease, disorder or condition caused by a nuclear receptor comprising a compound identified by the method of claim 1, wherein the compound comprises: formula:
(Chemical formula 3)
R ₁ —C═C— (C═O) —R ₂ —R ₃ or R ₁ — (C═O) —C═C—R ₂ —R ₃
Where R ₁ is an optionally substituted saturated or unsaturated hydrocarbon group having a maximum of 1 to 13 carbon atoms connecting the shortest distance, and R ₂ is A saturated or unsaturated hydrocarbon group which may be substituted, and R ₃ represents a substituent selected from the group consisting of hydrogen or an optionally substituted carbonyl group, hydroxyl group, amide group and thiol group; A composition in which R ₂ and R ₃ are monovalent substituents, and the maximum number of carbon atoms connecting the shortest distance is 4 to 14 with respect to the contained carbon. 1) A step of mixing a target biomolecule and a ligand to form a mixture;
2) adding fluorescently labeled maleimide to the mixture;
3) A method for quantifying the binding ability of a ligand to the target biomolecule, comprising detecting the binding between the target biomolecule and the fluorescently labeled maleimide in the mixture. 21. The method of claim 20, wherein the binding is a covalent bond, further comprising performing a quantification of the covalent bond, wherein the quantification is achieved by measuring at least two quantities for the ligand. The method according to claim 21, wherein the quantification of the covalent bond is performed using a ligand that is irreversibly covalently bound as a probe, wherein the binding of the ligand that is not covalently bound is quantified. 23. The method of claim 22, wherein the ligand comprises a covalent bond or a non-covalent bond. 21. The method of claim 20, wherein the biomolecule is a nuclear receptor. The biomolecule is a nuclear receptor, and the irreversibly covalently bound ligand is indomethacin, 15-deoxy-Δ ^12,14 -prostaglandin J ₂ (11-oxo-prosta-5Z, 9, 12E. , 14Z-tetraene-1-oic acid; 15d-PGJ ₂ ), 11-oxo-prosta-5Z, 12E, 14Z-triene-1-oic acid (CAY10410), 5-oxo-6E, 8Z, 11Z, 14Z- Eicosatetraenoic acid (5-oxoODE), 9-oxo-10E, 12Z-octadecanodienoic acid (9-oxoODE), 13-oxo-9Z, 11E-octadecanodienoic acid (13-oxoODE) ) And 15-oxo-5Z, 8Z, 11Z, 13E-eicososatetraenoic acid (5-oxoETE) It has the structure The method of claim 22. A system for identifying a compound that modulates a nuclear receptor, the system comprising:
A) a means for determining whether a candidate compound covalently binds to a cysteine in the nuclear receptor; and B) a candidate compound determined to be covalently bound is calculated to be a compound that modulates a nuclear receptor. Means to
A system comprising: A kit for measuring the covalent bond between a compound and the nuclear receptor, which is used to identify a compound that modulates the nuclear receptor,
A) a ligand known to be irreversibly covalently bound to the nuclear receptor; and B) instructions describing a procedure for measuring binding of a ligand that is not covalently bound;
A kit comprising: A program that causes a computer to execute a method of identifying a compound that modulates a nuclear receptor, the method comprising:
A) providing three-dimensional structure data of the candidate compound; and B) determining whether the candidate compound is covalently bound to cysteine in the nuclear receptor, the candidate determined to be covalently bound Identifying the compound as a compound that modulates a nuclear receptor,
Including the program. A recording medium storing a program for causing a computer to execute a method for identifying a compound that modulates a nuclear receptor, the method comprising:
A) providing three-dimensional structure data of the candidate compound; and B) determining whether the candidate compound is covalently bound to cysteine in the nuclear receptor, the candidate determined to be covalently bound Identifying the compound as a compound that modulates a nuclear receptor,
Including a recording medium. Indomethacin, 15-deoxy-Δ ^12,14 -prostaglandin J ₂ (11-oxo-prosta-5Z, 9,12E, 14Z-tetraene-1-oic acid; 15d-PGJ ₂ ), 11-oxo-prosta 5Z, 12E, 14Z-triene-1-oic acid (CAY10410), 5-oxo-6E, 8Z, 11Z, 14Z-eicosatetraenoic acid (5-oxoODE), 9-oxo-10E, 12Z-octadeca Nodienoic acid (9-oxoODE), 13-oxo-9Z, 11E-octadecanodienoic acid (13-oxoODE) and 15-oxo-5Z, 8Z, 11Z, 13E-eicososatetraenoic acid (5- A modulator of a nuclear receptor comprising a compound selected from the group consisting of oxo ETE). A method for determining whether a candidate compound is an agonist or antagonist of a nuclear receptor, the method comprising:
A) providing a candidate compound; and B) determining whether the candidate compound is covalently bound to a cysteine in the nuclear receptor and selecting the candidate compound determined to be covalently bound;
C) determining the activity of the candidate compound in a system comprising a nucleic acid construct comprising a nucleic acid sequence encoding a reporter operably linked to a transcription factor recognition sequence and the selected nuclear receptor. When the reporter expression is enhanced, the candidate compound is determined to be an agonist of the nuclear receptor, and when the reporter expression is decreased, the candidate compound is determined to be an antagonist of the nuclear receptor. , Process,
Including the method. 32. The method of claim 31, wherein the reporter is luciferase. 32. The method of claim 31, wherein the system is a cell.