Methods for analyzing the structure of complexes and their applications

Abstract

This provides a novel mechanism for structural analysis of complexes using NMR. [Solution] A method for analyzing the structure of a complex, comprising: obtaining a first transverse relaxation rate in an NMR measurement of the proton of a second molecule for a first sample containing a first molecule having an aromatic ring and a second molecule having a CH bond; obtaining a second transverse relaxation rate in an NMR measurement of the proton of a second molecule for a second sample containing the second molecule in a standalone state; and evaluating whether the first sample contains a hydrogen atom located near the aromatic ring in the first molecule based on the difference in transverse relaxation rates obtained by subtracting the second transverse relaxation rate from the first transverse relaxation rate.

Description

Technical Field

[0001] The present invention relates to a method for analyzing the structure of a complex and its use.

Background Art

[0002] In drug discovery research related to pharmaceuticals and agricultural chemicals, first, a hit compound that interacts with a target biomacromolecule, which is a target molecule, from a compound library and exhibits a drug effect is searched for, and synthetic development such as adding a modifying group to the hit compound is repeated to create a compound having desired physical properties and pharmacokinetics. With the development of recent computer technology and AI technology, in this series of drug discovery processes, research on a structure-based drug discovery method in which the structure of a complex of a target biomacromolecule and a drug candidate compound is analyzed and compound development is carried out based on this complex structure has been advanced.

[0003] In the structure-based drug discovery method, as methods for analyzing the three-dimensional structure of a complex of a target protein and a drug candidate compound, an X-ray crystal structure analysis method, a cryo-electron microscopy method, a nuclear magnetic resonance (NMR) method, etc. are known. In the X-ray crystal structure analysis method and the cryo-electron microscopy method, an electron density or density map of a complex of a low molecule and a high molecule is obtained, and structure modeling is performed on this electron density or density map. On the other hand, in the NMR method, for example, after obtaining intramolecular and intermolecular nuclear distance restrictions by a NOESY method or a transfer NOESY method using a nuclear Overhauser effect (NOE), a structure that satisfies the distance restrictions is calculated (see, for example, Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005] [Non-Patent Document 1] Molecules 25, 2974 (2020) [Non-Patent Document 2] Angew. Chem. 132, 14971-14978 (2020) [Non-Patent Document 3] ChemMedChem 25, e202300636 (2024) [Non-Patent Document 4] Methods Enzymol. 615, 177-236 (2019) [Non-Patent Document 5] J. Med. Chem. 59, 10788-10793 (2016) [Non-Patent Document 6] J. Med. Chem. 66, 10617-110627 (2023) [Non-Patent Document 7] Nat. Chem. Biol. 15, 822-829 (2019) [Overview of the project] [Problems that the invention aims to solve]

[0006] Incidentally, X-ray crystallography requires crystallization of the sample, while cryo-electron microscopy requires cooling and electron beam irradiation of the sample. In contrast, NMR has the advantage of enabling non-invasive analysis of samples under physiological solution conditions. Therefore, the application of NMR in the structural analysis of complexes is expected to expand.

[0007] This technology provides a novel mechanism for structural analysis of complexes using NMR. [Means for solving the problem]

[0008] To solve the above problems, for example, the configuration described in the claims may be adopted. This application includes several means for solving the above-mentioned problems, but one example is: To obtain the first transverse relaxation rate in the NMR measurement of the proton of the second molecule in a first sample containing a first molecule having an aromatic ring and a second molecule having a CH bond, To obtain the second transverse relaxation rate in the NMR measurement of the proton of the second molecule in a second sample containing the second molecule in a standalone state, Based on the difference in transverse relaxation rates obtained by subtracting the second transverse relaxation rate from the first transverse relaxation rate, it is evaluated whether the first sample contains hydrogen atoms located near the aromatic ring within the first molecule. Methods for analyzing the structure of a complex, including It is characterized by providing... [Effects of the Invention]

[0009] According to the present invention, a novel mechanism for structural analysis of complexes using NMR can be provided. Other issues, configurations, and effects not mentioned above will be clarified by the following description of the embodiments. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a schematic diagram illustrating the CH-π interaction between a CH bond and an aromatic ring. [Figure 2] Figure 2 is a schematic diagram illustrating the induced magnetic field around an aromatic ring under an external magnetic field. [Figure 3] Figure 3 is a schematic diagram of a complex structure analysis system using NMR according to one embodiment. [Figure 4] Figure 4 is a block diagram of a control device according to one embodiment. [Figure 5] Figure 5 is a flowchart illustrating the structural analysis of a complex using NMR according to one embodiment. [Figure 6] Figure 6 is a graph showing the difference in apparent transverse relaxation rate R2 (ΔR2) for each 1H of BI-9321 obtained in the example, both in its standalone state and when coupled to NSD3-PWWP. [Figure 7]Figure 7 shows the assignment of each 1H in the molecular structure of BI-9321 shown in Figure 6. [Figure 8] Figure 8 shows a method for setting the threshold value of ΔR2 for each 1H according to one embodiment. [Figure 9] Figure 9 shows a method for setting the threshold value of ΔR2 for each 1H according to another embodiment. [Figure 10] Figure 10 is a histogram of ΔR2 for each 1H of BI-9321 according to one embodiment. [Figure 11] Figure 11 shows the CH-π interaction between the aromatic amino acids in NSD3-PWWP and the 1H of BI-9321 in the complex prepared in the example. [Figure 12] Figure 12 shows the three-dimensional structure of the NSD3-PWWP and BI-9321 composite prepared in the example. [Figure 13] Figure 13 is a graph showing the distance between each 1H and aromatic amino acid in BI-9321 according to one embodiment. [Figure 14] Figure 14 is a graph showing the correlation between the distance to the aromatic ring of each 1H in BI-9321 according to one embodiment and ΔR2. [Figure 15] Figure 15 shows the three-dimensional structure of the NSD3-PWWP and BI-9321 complex based on complex data (PDB ID: 6G2O) obtained by X-ray crystallography. [Modes for carrying out the invention]

[0011] One of the initial challenges in structure-based drug discovery is finding molecules with strong binding affinity and interaction with target biomolecules. However, in reality, drug candidate molecules such as low-molecular-weight compounds and medium-sized molecules explored in the early stages of drug discovery often have low binding affinity to target macromolecules. There are limited methods for detecting interactions with low binding affinity; for example, X-ray crystallography and cryo-electron microscopy may not be able to determine the structure.

[0012] The inventors have discovered a simple and novel method for determining the structure of a complex with low interaction affinity, for example, by utilizing NMR, which can detect even low-affinity interactions with high sensitivity and reliability, and have filed this application.

[0013] Figure 1 is a schematic diagram illustrating the CH-π interaction. Figure 2 is a schematic diagram illustrating the induced magnetic field around the aromatic ring in the presence of an external magnetic field. For example, as shown in Figure 1, a weak attractive interaction caused by dispersion forces, known as the CH-π interaction, can act between adjacent CH bonds (CH) and aromatic rings (π electrons). Furthermore, as shown in Figure 2, the delocalized π electrons of the aromatic ring generate a ring current and an induced magnetic field when exposed to an external magnetic field (B0).

[0014] Here, when an external magnetic field is applied to the CH and aromatic rings forming the CH-π interaction, the direction of the induced magnetic field for hydrogen atoms located perpendicular to or near the aromatic ring plane becomes opposite to the direction of the external magnetic field (shielding effect). Therefore, the CH 1 The 1H NMR spectrum exhibits lower resonance frequencies (towards higher magnetic fields) compared to, for example, the case of a single compound (i.e., when no CH-π interaction is formed). Furthermore, the chemical shift value of the NMR spectrum decreases. Therefore, it can be seen that, for example, the CH-π interaction between a candidate drug compound and a target protein can be detected by changes in the chemical shift in the NMR spectrum (see, for example, Non-Patent Documents 2 and 3).

[0015] Next, consider a system in which a first molecule (P), such as a protein, and a second molecule (L), such as a candidate drug compound, interact in a 1:1 ratio to form a complex (PL), as described below. [ka]

[0016] The proportion of bound molecules existing as a complex (PL) in equilibrium is p BLet p be the ratio of the free-type molecules in which the first molecule (P) and the second molecule (L) are free. F Then, when an excess amount of the second molecule is present with respect to the first molecule (p F >> p B ), the apparent transverse relaxation rate R2 of the compound NMR signal is represented by the following formula (1).

[0017]

Equation

[0018] Here, in formula (1), ·R 2,F and R 2,B are respectively the (true) transverse relaxation rates of the free-type molecules and the bound-type molecules, ·k ex = k on satisfies [P] + k off , k on is the association rate constant, k off is the dissociation rate constant, <00​​​​​​​​​​​​​​​​​​​​​​​​As this increases, the second molecule (L) forms a CH-π interaction when it binds to the first molecule (P). 1 The apparent transverse relaxation rate R2 of H is not forming an interaction with other 1 It can be seen that this is larger than the apparent transverse relaxation velocity R2 of H.

[0021] Furthermore, it is known that in systems where the single state and the bound state are in equilibrium, such as the interaction between the first molecule (P) and the second molecule (L), if there is a difference in the changes in the chemical shifts between the single state and the bound state, the apparent transverse relaxation rate R2, which can be measured using NMR, becomes larger (Non-Patent Literature 4). As mentioned above, this change in the apparent transverse relaxation rate R2 is influenced not only by the difference in chemical shifts between the equilibrium states, but also by the abundance ratio and the equilibrium dissociation rate constant k. on , k off or equilibrium dissociation constant K d It also depends on this change in R2. off Ya K d Calculate k off Ya K d Based on this, methods for quantitatively evaluating the interaction between a first molecule (P) and a second molecule (L) have been proposed (see, for example, Patent Document 1 and Non-Patent Documents 5-6).

[0022] Here, the inventors conceived of obtaining structural information of the complex by utilizing the difference in transverse relaxation rate R2, since the apparent change in transverse relaxation rate R2 can be calculated based on the NMR spectrum obtained by NMR measurement, and the protons interacting for the formation of the complex can be identified.

[0023] In other words, the second molecule (L) is in a standalone state (p B If R² = R (=0), then R² = R 2,F Therefore, regarding the state of the second molecule (L) alone and the state in which it is bound to the first molecule (P), the intramolecular structure 1By directly measuring the change in the apparent transverse relaxation rate R2 of H based on NMR measurements and comparing these values, it is possible to identify the atomic nuclei within the second molecule (L) that are interacting (i.e., bound) to the first molecule (P). For example, specifically, multiple nuclei contained within the second molecule (L) 1 Of the H atoms, one forms a CH-π interaction with the aromatic ring within the first molecule (P). 1 H can be identified.

[0024] Therefore, if the atomic nuclei that form such low-affinity interactions (interaction sites) can be identified by detecting changes in their chemical shifts using NMR spectroscopy, their positions within the molecule can be determined. Furthermore, if the interacting sites can be identified in the first molecule (P) and the second molecule (L), the three-dimensional structure of the complex composed of these molecules can be estimated by sterically combining them so that their interacting sites are in close proximity to each other. This technology is based on these findings.

[0025] The following explanation of this technology will be based on an example of structural analysis of a composite according to one embodiment, with reference to the drawings as appropriate. Note that in each drawing, components having the same function may not be denoted by reference numerals or described again.

[0026] [Structural Analysis System] The structural analysis system for the complex according to this technology is a system that performs structural analysis of the complex using nuclear magnetic resonance. This analysis system is not limited to this, but it can suitably perform the structural analysis method for the complex according to this technology. Figure 3 is a schematic diagram of the structural analysis system 100 for the complex using NMR according to one embodiment. Figure 4 is a block diagram of the control device 103 according to one embodiment.

[0027] The structural analysis system 100 generally comprises one or more magnet units 101, one or more spectrometers 102, one or more control devices 103, and one or more databases 104. The structural analysis system 100 may additionally include one or more user terminals 105. Each of these elements is configured to send and receive information from one another, for example, via a wired or wireless network. Two or more of these elements may be configured as a single unit. Alternatively, these elements (in particular the control devices 103 and the databases 104, etc.) may each be configured as two or more separate units.

[0028] While not limited to this, a typical example is that the magnet unit 101 is connected to the spectrometer 102 and the control device 103 via wires. The spectrometer 102 and the control device 103 are connected via wires or wirelessly. The control device 103 and the database 104 are connected via wires or wirelessly. The magnet unit 101, the spectrometer 102, and the control device 103 can be combined to form an NMR apparatus. A commercially available NMR apparatus can be used without any particular restrictions.

[0029] The control device 103 may also store (integrate) part or all of the database 104 internally. The user terminal 105 is configured to connect to the control device 103, for example, via wireless communication.

[0030] The magnet unit 101 is a device for applying a magnetic field to a sample. The magnet unit 101 includes, for example, a magnet unit for applying a magnetic field to the sample and a housing for housing the sample and the probe unit of the spectrometer 102, which will be described later. The magnet unit typically includes a superconducting magnet. The magnet unit 101 may also include, for example, a shim coil for adjusting the uniformity of the magnetic field, a locking system for correcting magnetic field drift, a gradient magnetic field coil, and the like.

[0031] The spectrometer 102 is a device that performs the excitation of a sample and the detection of an NMR signal. The spectrometer 102 generally comprises a high-frequency generation unit, a probe unit, and a receiving unit. The high-frequency generation unit includes, for example, an oscillator for generating electromagnetic waves (which may be radio waves) to excite atomic nuclei in the sample, and transmits the generated radio waves to the probe unit. The probe unit is housed inside the magnet unit 101. The probe unit includes, for example, an RF coil for irradiating the sample with radio waves and detecting an NMR signal. The receiving unit includes, for example, a duplexer for extracting the NMR signal from the signal detected by the RF coil, an amplifier for amplifying the extracted NMR signal, a phase detector for detection, memory, a control unit for controlling these, etc.

[0032] The control device 103 is an element that controls the operation of the magnet unit 101 and the spectrometer 102 (i.e., the operation of the NMR measuring device). The control device 103 is also configured to perform structural analysis of the complex based on the results of the NMR measurement.

[0033] Figure 4 is a block diagram showing the configuration of a control device 103 according to one embodiment. The control device 103 optionally includes, for example, a central processing unit (CPU 51) that performs digital signal processing and various programs, a main memory such as a ROM (read-only memory) that stores programs executed by the CPU 51 (an example of a processor) and a RAM (random access memory) used as a working area for expanding programs, an auxiliary storage device such as flash memory or EEP-ROM (Electrically Erasable Programmable Read-Only Memory), and an input / output unit (IF) 53 that sends and receives various information with the magnet unit 101, spectrometer 102, database 104, etc. In Figure 4, the main memory and auxiliary storage device together are shown as memory 52.

[0034] Furthermore, the control device 103 includes a measurement unit 54, an acquisition unit 55, an evaluation unit 56, and a structure estimation unit 57. Each of these parts of the control device 103 may be composed of hardware such as circuits, functionally realized by the CPU 51 executing a computer program, or realized by the cooperation of hardware and software. In this embodiment, each functional element of the control device 103 is realized by the CPU 51 executing these programs and applications stored in the main memory.

[0035] The measurement unit 54 controls the operation of the magnet unit 101 and the spectrometer 102 to perform NMR measurements. The measurement unit 54 can, for example, set NMR measurement conditions. The measurement unit 54 can, for example, drive the magnet unit 101 to generate a uniform magnetic field of a predetermined magnetic field strength. The measurement unit 54 can, for example, generate radio waves with the spectrometer 102 and irradiate the sample via the RF coil. The measurement unit 54 can, for example, control the frequency, timing, etc., of the radio waves generated by the spectrometer 102. The measurement unit 54 can, for example, detect the induced current generated in the RF coil with the spectrometer 102 and record it as digital information (NMR signal). The NMR signal can be stored, for example, in the memory of the spectrometer 102 or the memory 52 of the control device 103.

[0036] The acquisition unit 55 acquires information necessary for evaluation and structural analysis. The acquisition unit 55 acquires information necessary for evaluation and structural analysis from, for example, the memory of the spectrometer 102, the memory 52 of the control device 103, the database 104, etc.

[0037] The evaluation unit 56 evaluates the molecules using the NMR measurement results. For example, the evaluation unit 56 evaluates whether the first sample, which includes the first molecule and the second molecule to be evaluated, contains hydrogen atoms located near the aromatic ring in the first molecule. The details of the evaluation performed by the evaluation unit 56 will be described later.

[0038] The structure estimation unit 57 estimates the structure of a molecule using NMR measurement results. Based on, for example, the position information of the aromatic ring in the three-dimensional structure data of the first molecule and the position information of hydrogen atoms in the three-dimensional structure data of the second molecule that have been identified as being located near the aromatic ring in the first molecule, the structure estimation unit 57 estimates the position and angle of the second molecule that is bound to the first molecule. The structure estimation unit 57 in this embodiment has the function of displaying, analyzing, and simulating (for example, molecular dynamics calculations; the same applies hereinafter) the three-dimensional structure of proteins, DNA, etc. The structure estimation unit 57 may be configured to realize the display and analysis function in cooperation with an external device that has the function of displaying, analyzing, and simulating the three-dimensional structure of proteins, DNA, etc. An example of software for performing molecular dynamics calculations is so-called MD software such as GROMACS.

[0039] Database 104 stores various data necessary for using the structural analysis system 100. Database 104 stores, but is not limited to, the following information: • Regarding the second molecule (in its standalone state) 1 Assignment information for each proton in the 1H NMR spectrum (correspondence information with 3D stereostructure, chemical structure, etc.) • Three-dimensional structural data of the first molecule (in isolation) • Three-dimensional structural data of the second molecule (in isolation) • Structural data of the complex consisting of the first and second molecules Furthermore, the first and second molecules can preferably be those described later.

[0040] Furthermore, the structural analysis system 100 may be linked to an external database (for example, a protein database, etc., not shown). The acquisition unit 55 and structural estimation unit 57 of the control device 103 may also be configured to acquire necessary information from an external database.

[0041] The user terminal 105 is a terminal operated by a user utilizing the structural analysis system 100, and consists of devices such as a smartphone, tablet, notebook PC, or desktop PC. The user terminal 105 may have the same configuration and / or functions as some or all of the control device 103 described above. The user terminal 105 has the same configuration and / or functions as the measurement unit 54, acquisition unit 55, evaluation unit 56, and structural estimation unit 57 of the control device 103. Further explanation of each of these units is omitted. The user can, for example, perform operations such as setting the operation of the NMR instrument, controlling its operation, and utilizing measurement results (evaluation and analysis, etc.) from a remote location away from the NMR instrument via the user terminal 105.

[0042] [Structural analysis method] The structural analysis method for complexes related to this technology utilizes nuclear magnetic resonance to analyze the structure of complexes. This technology can target complexes in which a first molecule and a second molecule are formed by low-affinity interactions. In other words, it can detect whether the first molecule and the second molecule are bound by low-affinity interactions. Furthermore, for complexes composed of such low-affinity interactions, the binding structure of the first molecule and the second molecule (e.g., the position and orientation (angle) of the bond) can be analyzed.

[0043] The sample to be evaluated by nuclear magnetic resonance (NMR) is typically a solution sample. For example, a solution sample containing the first molecule and the second molecule can be designated as the first sample. A solution sample containing the second molecule in a standalone state can be designated as the second sample. "The second molecule in a standalone state" means that it does not form an interaction (is not bound) to the first molecule. Within the limits that do not impair the function and effect of this technology, the first sample may contain other components besides the first and second molecules. Similarly, within the limits that do not impair the function and effect of this technology, the second sample may contain other components besides the second molecule.

[0044] The interaction between the first and second molecules can be evaluated if it has a low binding affinity. While such low-binding-affinity interactions are not strictly limited, for example, the dissociation constant (K) d High values ​​of the dissociation constant (K) can be considered, for example, d Examples of interactions in systems where the dissociation constant (K) is 50 mM or higher, 10 mM or higher, 1 mM or higher, and 100 μM or higher are given. d ) is the ratio of the dissociation rate constant to the association rate constant: k off / k on It can be expressed as ;. Examples of such interactions with low binding affinity (in other words, high dissociation rate constants) include CH-π interactions and hydrophobic interactions (hydrophobic effects).

[0045] The first and second molecules constituting the complex are not particularly limited, and for example, combinations of molecules that form interactions with low binding affinity can be targeted. In one embodiment, the first molecule can be, for example, a protein, nucleic acid, or any biomolecule that can be a drug target for pharmaceuticals and agrochemicals. In another embodiment, the second molecule can be, for example, various molecules that can act as a ligand or drug candidate compound for the first molecule. This allows, for example, the second molecule to be screened as a hit compound when the first and second molecules form an interaction.

[0046] In one embodiment, the first molecule can be a macromolecule such as a protein that can serve as a target for drug discovery, such as pharmaceuticals and agrochemicals. In another embodiment, the second molecule can be a so-called small molecule or medium molecule compound that can serve as a ligand or drug candidate molecule for the first molecule. By using such a combination of the first and second molecules, the medium to small molecule compound can more easily enter cells containing macromolecules, increasing the likelihood of binding to finer targets.

[0047] More specifically, the first molecule can typically have a molecular weight of 10,000 or more, for example, 15,000 or more, 50,000 or more, or 100,000 or more. The second molecule can be a medium- or low-molecular-weight molecule with a relatively smaller molecular weight than the first molecule, for example, typically 10,000 or less, for example, 5,000 or less, preferably 3,000 or less, and as an example, about 150 to 3,000. In one embodiment, the molecular weight of the first molecule is preferably greater than the molecular weight of the second molecule, typically twice or more the molecular weight of the second molecule, for example, three times or more, five times or more, or ten times or more.

[0048] In one embodiment, when the interaction with low binding affinity is a CH-π interaction, the first molecule is exemplified as a molecule having an aromatic ring, and the second molecule as a molecule having a CH bond. Although not limited thereto, the aromatic ring of the first molecule is preferably derived from, for example, an aromatic amino acid moiety. The aromatic amino acid moiety can be, for example, phenylalanine (molecular weight: 165.19), tryptophan (molecular weight: 204.23), histidine (molecular weight: 155.1546), and tyrosine (molecular weight: 181.19). Since these aromatic amino acids are protein-constituting amino acids, molecules containing such aromatic amino acid moieties can be suitably considered as the first molecule.

[0049] Furthermore, in order to reliably form a complex between the first and second molecules, which form an interaction with low binding affinity, when obtaining the first transverse relaxation rate as described later, it is desirable to sufficiently increase the abundance of the second molecule compared to the first molecule in the first sample used for NMR measurement to obtain the first transverse relaxation rate. Specifically, for example, in the solution sample used for NMR measurement, it is desirable to increase the molar concentration of the second molecule compared to the molar concentration of the first molecule. This cannot be said definitively as it depends on the structures of the first and second molecules, but for example, the molar concentration of the second molecule is preferably three times or more than the molar concentration of the first molecule, and more preferably five times or more, ten times or more, fifty times or more, one hundred times or more, five hundred times or more, one thousand times or more, and one thousand times or more, and five thousand times or more.

[0050] In the following section, we will describe how to evaluate the presence or absence of binding in a complex and how to analyze the binding structure, using the structural analysis system 100, in the case where the first molecule and the second molecule form a complex through CH-π interaction.

[0051] Figure 5 is a flowchart illustrating the structural analysis of a complex using NMR according to one embodiment. The structural analysis method of this embodiment generally includes evaluation steps for the presence or absence of molecular bonding, shown as steps 501 to 504 and 509 in Figure 5, and structural analysis steps for the complex, shown as steps 505 to 508. The evaluation steps and structural analysis steps can each be performed independently. Hereinafter, for example, "step 501" will be denoted as "S501".

[0052] In the evaluation step, for example, it is evaluated whether the first molecule and the second molecule form a complex. The evaluation step generally includes a step of obtaining the first transverse relaxation rate (S501), a step of obtaining the second transverse relaxation rate (S502), a step of determining the difference in transverse relaxation rates (S503), and a step of evaluating the molecules (S504).

[0053] In the first transverse relaxation rate acquisition step, for example, for a first sample containing a first molecule having an aromatic ring and a second molecule having a CH bond (an example of an interaction site), the first transverse relaxation rate is acquired by NMR measurement of the proton of the second molecule (S501). The aromatic ring of the first molecule and the CH bond (more specifically, the proton) of the second molecule are examples of interaction sites in each molecule.

[0054] In the second transverse relaxation rate acquisition step, the second transverse relaxation rate is acquired for a second sample containing the second molecule in a standalone state by measuring the protons of the second molecule using NMR (S502). The first and second transverse relaxation rates can be calculated by the acquisition unit 55 based on the results of the NMR measurement performed by the measurement unit 54 of the control device 103, which operates the magnet unit 101 and the spectrometer 102 to perform NMR measurements of protons. Since the transverse relaxation rate R2 [1 / s] is the reciprocal of the transverse relaxation time T2 [s], acquiring the first and second transverse relaxation rates is equivalent to acquiring the first and second transverse relaxation times.

[0055] In one embodiment, for example, the measurement unit 54 outputs an instruction to the magnet unit 101 and the spectrometer 102 to measure the NMR signal of a second molecule by proton NMR measurement according to the CPMG (Carr-Purcell-Meiboom-Gill) method. In the CPMG method, for example, the magnetization of the sample is fixed on the xy plane of a rotating coordinate system by repeatedly applying 180° pulses of radio waves to the sample in a magnetic field at regular intervals to obtain a FID (Free Induction Attenuation) signal.

[0056] Next, the acquisition unit 55, for example, uses the spectrometer 102 to determine the second molecule based on the acquired NMR signal. 1 The unit outputs an instruction to generate an H NMR spectrum. Then, the acquisition unit 55 outputs, for example, the generated second molecule 1 The transverse relaxation rate is calculated based on the 1H NMR spectrum. The method for calculating the transverse relaxation rate will be specifically explained in the examples described later.

[0057] The acquisition unit 55 stores the calculated transverse relaxation rate for the first sample containing the first molecule and the second molecule in the memory 52, for example, as the first transverse relaxation rate. The acquisition unit 55 also stores the calculated transverse relaxation rate for the second sample in which the second molecule is in a standalone state in the memory 52, for example, as the second transverse relaxation rate.

[0058] Furthermore, if at least one of the first lateral relaxation speed and the second lateral relaxation speed is pre-stored in memory 52 or the like, the acquisition unit 55 may acquire the first and / or second lateral relaxation speed by reading the first and / or second lateral relaxation speed stored in memory 52 or the like. Alternatively, the acquisition unit 55 may acquire the first and / or second lateral relaxation speed by reading the first and / or second lateral relaxation speed from, for example, another device or database inside or outside the system.

[0059] In the determination step, the evaluation unit 56 determines whether the change in the transverse relaxation rate, that is, the difference in the transverse relaxation rate ΔR2 obtained by subtracting the second transverse relaxation rate from the first transverse relaxation rate obtained, is greater than or equal to a predetermined threshold (S503). The predetermined threshold is the difference in chemical shift due to the formation of a complex between the first molecule and the second molecule by an interaction with low affinity (for example, 1 The value can be adjusted to a level where changes in the chemical shift of the 1H NMR spectrum can be detected.

[0060] (1) Specifically, the predetermined threshold is, for example, 0.05S -1 It can be set to the above, for example, 0.1S -1 More than 0.2S -1 More than 0.3S -1 More than 0.4S -1 The above is 0.5S -1 The above may be the case. (2) The predetermined threshold can also be, for example, the sum of the mean and standard deviations (mean+1σ) of the difference in transverse relaxation rates ΔR2 for all protons of the second molecule (see Figure 8). (3) Alternatively, the predetermined threshold can be, for example, the third quartile (also called the 75th percentile) of the difference ΔR2 in the transverse relaxation rates of all protons in the second molecule (see Figure 9). A percentile is the value that is located at the 100 × αth position when the data is arranged in ascending order (where 0 ≤ α ≤ 1). In addition to 75, any other percentile value such as 65, 70, 80, 85, 90, etc. (100 × α, for example 0.6 ≤ α ≤ 1) can be used.

[0061] Then, if the difference ΔR2 between the first transverse relaxation rate and the second transverse relaxation rate is greater than or equal to a predetermined threshold (YES in S503), the evaluation unit 56 evaluates that the second molecule contains a proton located near the aromatic ring of the first molecule (S504). Here, "nearby" can be understood as a distance at which the second molecule forms an interaction with the first molecule that has low affinity (e.g., a CH-π interaction). Alternatively, "nearby" can be defined as a distance at which the second molecule forms a complex with the first molecule. As a specific example, "nearby" means that the distance between the aromatic ring in the first molecule and the hydrogen atom (proton) of the CH bond in the second molecule is within 6 Å. The distance between the aromatic ring in the first molecule and the hydrogen atom (proton) of the CH bond in the second molecule is preferably within 5 Å, and more preferably within 4 Å, 3 Å, etc.

[0062] From the above, the evaluation unit 56 can evaluate, for example, that the second molecule forms a complex with the first molecule when the difference in transverse relaxation rates ΔR2 is greater than or equal to a predetermined threshold. Alternatively, the evaluation unit 56 can evaluate, for example, that the second molecule forms a low-affinity interaction (e.g., a CH-π interaction) with the first molecule when the difference in transverse relaxation rates ΔR2 is greater than or equal to a predetermined threshold. Alternatively, the evaluation unit 56 can evaluate, for example, that a hydrogen atom of the second molecule is positioned near an aromatic ring in the first molecule when the difference in transverse relaxation rates ΔR2 is greater than or equal to a predetermined threshold.

[0063] The evaluation unit 56 can, from another perspective, measure the number of atoms in the second molecule that form low affinity interactions with the first molecule by measuring the difference ΔR2 between the first transverse relaxation rate and the second transverse relaxation rate. For example, by measuring the difference ΔR2 between the transverse relaxation rates, the evaluation unit 56 can measure the number of protons in the second molecule located near the aromatic ring in the first molecule. If the difference ΔR2 between the first transverse relaxation rate and the second transverse relaxation rate is greater than or equal to a predetermined threshold, the evaluation unit 56 can determine that, for example, the number of hydrogen atoms in the second molecule (e.g., hydrogen atoms) whose difference exceeds the predetermined threshold is equal to the number of hydrogen atoms in the second molecule located near the aromatic ring in the first molecule.

[0064] On the other hand, if the difference ΔR2 between the first transverse relaxation rate and the second transverse relaxation rate is not greater than or equal to a predetermined threshold (NO in S503), the evaluation unit 56 evaluates that the second molecule does not contain a proton located near the aromatic ring of the first molecule (S509). In other words, the evaluation unit 56 can evaluate, for example, that the second molecule does not form a complex with the first molecule. Alternatively, the evaluation unit 56 can evaluate, for example, that the second molecule does not form a low-affinity interaction with the first molecule.

[0065] In the structural analysis process, for example, the three-dimensional structure (binding structure) of the complex of the first molecule and the second molecule is estimated. The structural analysis process generally involves the second molecule 1 The process includes obtaining proton assignment information from the 1H NMR spectrum (S505), identifying hydrogen atoms in the second molecule (S506), obtaining three-dimensional structural data of the first and second molecules (S507), and estimating the three-dimensional structure of the complex (S508). Each of these steps can be performed by the structure estimation unit 57 of the control device 103, for example, following S504 or based on instructions from the user.

[0066] The structure estimation unit 57, for example, from the database 104, determines the second molecule in its standalone state. 1Obtain proton assignment information for the 1H NMR spectrum (S505). Assignment information refers to the protons of the second molecule in its standalone state. 1 H NMR signal (for example, 1 The 1H NMR spectrum (part or all of it) provides information indicating which proton in the three-dimensional structure or chemical structure of the second molecule it corresponds to.

[0067] Then, the structure estimation unit 57 identifies, based on the acquired attribution information and the evaluation results from S504, which part of the three-dimensional stereostructure or chemical structural formula of the second molecule corresponds to the interaction site (e.g., proton) in the second molecule that was evaluated to be located near the interaction site (e.g., aromatic ring) in the first molecule (S506).

[0068] Next, the structure estimation unit 57 obtains, for example, the three-dimensional structure data of the first molecule and the three-dimensional structure data of the second molecule from the database 104 (S507). Then, the structure estimation unit 57 combines the first molecule and the second molecule such that the interaction sites in the first molecule (e.g., aromatic rings) and the interaction sites in the second molecule (e.g., protons) are located in close proximity to each other. As a result, the structure estimation unit 57 can estimate the three-dimensional structure of the complex consisting of the first molecule and the second molecule (S508).

[0069] Furthermore, with respect to the protons in the second molecule, the larger the difference in transverse relaxation rates ΔR2, the closer they are to the aromatic ring (interaction site) of the first molecule. The difference in transverse relaxation rates ΔR2 and the distance between the interaction sites can, for example, have a linear relationship. Therefore, the structure estimation unit 57 may be configured to calculate the distance of the protons in the second molecule from the aromatic ring (interaction site) in the first molecule based on the difference in transverse relaxation rates ΔR2, set a predetermined threshold for distance to distinguish between the distance of protons evaluated as being located near the aromatic ring (interaction site) in the first molecule and the distance of protons evaluated as not being located near the aromatic ring (interaction site) in the first molecule, and estimate the arrangement of the first and second molecules in the complex on the condition that the distance of protons evaluated as being located near the aromatic ring (interaction site) in the first molecule from the aromatic ring (interaction site) in the first molecule is within this predetermined threshold. The predetermined threshold for distance may be the distance between the maximum distance for a proton evaluated as being located near the aromatic ring (interaction site) in the first molecule and the minimum distance for a proton evaluated as not being located near the aromatic ring (interaction site) in the first molecule, and can be, for example, the midpoint between the above maximum and minimum values.

[0070] According to this technology, the formation of the complex and the three-dimensional structure of the complex can be evaluated and estimated by simple NMR measurements. Furthermore, this technology allows for simple one-dimensional NMR measurements. 1 One advantage is that only H transverse relaxation measurements are required. In addition, this technology eliminates the need to introduce stable isotopes into the first molecule, and furthermore, the amount of the first molecule required can be kept to a minimum.

[0071] (Examples) This technology was used to test whether it is possible to identify whether a ligand, as a second molecule, is bound to a predetermined binding site on a protein, as a first molecule. As the evaluation sample in this example, a complex of the human protein NSD3 and BI-9321, which selectively binds to the PWWP1 domain, as reported in Non-Patent Literature 1, was used.

[0072] 1. Protein sample Protein samples were prepared by making the following modifications to the PWWP domain of NSD3, a type of histone lysine methyltransferase (amino acid numbers 247-398 in NSD3; molecular weight 18000; hereinafter sometimes simply referred to as "NSD3-PWWP").

[0073] First, E. coli BL21 (DE3) was transformed with a cold-shock-inducible plasmid vector containing a His×6 tag, glutathione S-transferase (GST), TEV protease recognition sequence, HRV3C protease recognition sequence, and DNA sequence encoding NSD3-PWWP. The transformed E. coli were then grown in LB medium (10 mL) at 37°C for 15.5 hours. Next, the grown E. coli were transferred to LB medium (2 L) and cultured at 37°C until the OD600 reached 0.9. Isopropyl β-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.3 mM to induce NSD3-PWWP synthesis. After IPTG addition, the cells were cultured at 15°C for another 15.5 hours and then centrifuged.

[0074] The obtained bacterial cells were purified using a known method similar to that described in Non-Patent Document 7 to obtain a protein sample. For cleaving the protein attached to the N-terminus of NSD3-PWWP, the method described in Non-Patent Document 7 was modified, and HRV3C protease was used. After preparing NSD3-PWWP, the lysis buffer was replaced with 99% D2O, 20 mM phosphate buffer (pH 7.0), and 100 mM NaCl.

[0075] 2.Low molecular compounds BI-9321 (Medchemexpress, molecular weight 360.43), an NSD3-PWWP ligand, was dissolved in DMSO-d6, a deuterated NMR solvent. The structural formula of BI-9321 used is shown below.

[0076] [ka]

[0077] 3. Measurement sample Two samples were prepared for NMR measurement: one with a concentration of (low molecular weight) BI-9321 at 1 mM, and one with (high molecular weight) NSD3-PWWP (final concentration 10 μM) added (Sample 1), and one without (Sample 2). The solvent for the measurement samples was 20 mM phosphate buffer (pH 7.0), 100 mM NaCl, 94% D2O, and 5% DMSO-d6, and the samples were sealed in φ5 mm sample tubes for SampleJet (Bruker).

[0078] 4.NMR measurement conditions The two prepared measurement samples are placed in the autosampler of the NMR spectrometer, and the measurement unit 54 of the control device 103 specifies the measurement conditions, thereby enabling each measurement sample to be measured by the BI-9321 1 The transverse relaxation rate R2 of the H nucleus was measured. The CPMG method was used to measure the transverse relaxation rate R2. The details of the measurement conditions are as follows.

[0079] • Equipment: Avance Neo 800 (Bruker) • Probe: TCI cryoprepole (Bruker) ·Measurement temperature: 298K • Pulse sequence: CPMG multi-echo sequence with presaturation inserted to remove residual solvent signal. • Delay time before and after the 180° pulse of the CPMG-sequenced spin echo: 1 millisecond Spin echo count: 4, 8, 10, 20, 40, 50, 100, 150, 250, 350, 500, 1000 • Repeat time: 6 seconds • Presaturation time: 4 seconds • Total count: 16 • Measurement time: 1 hour 33 minutes 44 seconds

[0080] 5. NMR Data Analysis The measurement unit 54 of the control device 103 performs a Fourier transform on the obtained NMR signal (FID signal) for each wavenumber component, 1 The 1H NMR spectrum was prepared. Then, the acquisition unit 55 of the control device 103 obtained 1 From the 1H NMR spectrum, BI-9321 1 The lateral relaxation rate R2 was calculated by calculating the integral value (i.e., area) of each H signal and fitting its change with relaxation time t to the theoretical formula: I(t) = I0·exp(-R2t). In the above equation, I0 is the integral value when the relaxation time is set to 0.

[0081] The evaluation unit 56 evaluates each of the BI-9321 1 For the H nucleus, the difference ΔR2 between the transverse relaxation rate R2 of measurement sample 1 and the transverse relaxation rate R2 of measurement sample 2 was calculated. Figure 6 shows the calculated BI-9321 1 The difference ΔR2 (increment) in the lateral relaxation rate R2 for each H is shown. Figure 7 shows the two-dimensional structural formula of BI-9321, as shown in Figure 6. 1 The assignment of the H signal was shown.

[0082] Figure 8 shows each of the embodiments 1 This figure illustrates the setting of the threshold value (sum of mean and standard deviation) for ΔR² of H. The evaluation unit 56 evaluates each of the BI-9321 1 For ΔR2 of H, the sum of the mean and standard deviation of ΔR2 (for example, slightly over 0.3) was set as the criterion (threshold) for the increase in ΔR2 due to coupling with NSD3-PWWP. Then, the evaluation unit 56 uses this threshold as a reference to evaluate each of the BI-9321 1 The evaluation checked whether ΔR2 of H was greater than or equal to a threshold. As a result, the evaluation unit 56 was identified as 1 to 12 in BI-9321. 1 Of H, 1It was confirmed that the ΔR2 values ​​for H3, 6, and 10 were above the threshold, indicating an increase in ΔR2.

[0083] Figure 10 shows each of the BI-9321 according to one embodiment. 1 This is a histogram of ΔR2 for H. The ΔR2 histogram shows bimodality, and it can be confirmed that it can be neatly divided into two groups by a threshold of slightly over 0.3. 1 When H is divided into two groups based on the above threshold for ΔR2 and a t-test is performed, the p-value is 8.9 × 10⁻⁶. -5 And so, other 1 Compared to H 1 A significant increase in ΔR2 was observed for H3, H6, and H10. Furthermore, as shown in Figure 9, a significant increase in ΔR2 can be evaluated even when the third quartile of ΔR2 is used as the threshold.

[0084] Based on the above, the evaluation unit 56 made the following evaluation, for example. BI-9321 contains a hydrogen atom located near the aromatic ring of NSD3-PWWP. The hydrogen atom in BI-9321 is 1 There are three types: H3, H6, and H10. · BI-9321 1 H3,6,10 form low-affinity interactions with NSD3-PWWP.

[0085] Next, based on the estimation by the evaluation unit 56, the structure estimation unit 57 obtains the 3D structure data of NSD3-PWWP and the 3D structure data of BI-9321 from the database 104, and the BI-9321 1 The H3,6,10 positions were evaluated to determine if they were in a relationship where they could interact with NSD3-PWWP. The structure estimation unit 57 obtained crystal structure information of "BI-9321" and "NSD3-PWWP" from the Protein Data Bank (PDB) using X-ray diffraction. Furthermore, the structure estimation unit 57 used its display and analysis function to determine the structure of BI-9321 based on this acquired data. 1We confirmed that H3, 6, and 10 are in a configuration that allows for CH-π interactions with the aromatic amino acids of NSD3-PWWP.

[0086] Specifically, for example, the structural estimation unit 57 first determines each of the BI-9321 based on R2. 1 The distance of H from the aromatic amino acid (aromatic ring) was calculated. Figure 13 shows each of the BI-9321 according to one embodiment. 1 This is a graph showing the distance between H and aromatic amino acids, and Figure 14 shows each of BI-9321 according to one embodiment. 1 This graph shows the correlation between the distance to the aromatic ring of H and ΔR2. The regression line in Figure 14 was calculated using the linear least squares method with the lingress function in SciPy. 1 A certain correlation is observed between the distance to the aromatic ring of H and ΔR2, for example, 1 It can be observed that the smaller the distance between H and the aromatic ring, the larger the ΔR2 tends to be.

[0087] The structural estimation unit 57 then proceeds, 1 Maximum distance for H3, 6, 10 ( 1 H6 (3.63 Å) and the remaining 1 Minimum distance for H ( 1 A value between H5 (3.74 Å) and (for example, 3.60 Å) was set as the distance threshold (distance condition). Then, the structure estimation unit 57 used molecular dynamics simulation function to determine the BI-9321 1 The relative configuration of NSD3-PWWP and BI-9321 was searched such that H3, 5, 6, and 10 are located within 3.60 Å of the aromatic amino acid (aromatic ring) of NSD3-PWWP, and a configuration satisfying this distance condition was found. The structural estimation unit 57 estimated the three-dimensional structure resulting from this arrangement of NSD3-PWWP and BI-9321 to be the three-dimensional structure of the NSD3-PWWP and BI-9321 complex.

[0088] Figure 11 shows the aromatic amino acids in NSD3-PWWP and BI-9321 of the complex prepared in the example. 1This figure shows the CH-π interaction between H atoms and the three-dimensional structure of the complex. Figure 12 shows the three-dimensional structure of the NSD3-PWWP and BI-9321 complex prepared in the example. The structural estimation unit 57, for example, places the aromatic ring of the aromatic amino acid of NSD3-PWWP near the aromatic ring of BI-9321. 1 By arranging H3, 6, and 10, the three-dimensional structure of the complex formed by the CH-π interaction between BI-9321 and NSD3-PWWP was estimated, as shown in Figures 11 and 12. As shown in these figures, it was confirmed that the structure estimation unit 57 can visually display how the ligand compound consisting of BI-9321 is bound to the NSD3-PWWP protein.

[0089] The structural estimation unit 57, for example, calculates the binding sites, binding distances, and binding angles of both molecules based on the estimated three-dimensional structure of the complex, and stores this structural information in the memory 52.

[0090] For reference, Figure 15 shows the three-dimensional structure of the complex based on X-ray diffraction crystal structure data of "protein molecule NSD3-PWWP that forms a complex with the small molecule compound BI-9321," recorded in the Protein Data Bank (PDB) as (PDB ID: 6G2O). From this three-dimensional structure, we can see that BI-9321 1 It can be confirmed that H3, 6, and 10 can form a complex through CH-π interactions with the aromatic rings of the aromatic amino acids in NSD3-PWWP.

[0091] From the above, protons in low molecular weight compounds ( 1 Of the H) compounds, those that form CH-π interactions with aromatic amino acids in proteins can be identified from changes in the transverse relaxation rate R2. Confirmation of CH-π interactions and structural analysis can be performed using the structural analysis system 100 related to this technology.

[0092] For example, Non-Patent Document 3 discusses the low molecular weight compounds in the complex. 1To directly measure the H chemical shift, deuterium is introduced into the protein, and the protein concentration in the complex solution is set to 200 μM for two-dimensional NMR measurement. However, the method according to this technology does not require deuterium labeling of the protein. Furthermore, the protein concentration in the sample only needs to be around 10-50 μM, which is 1 / 20 to 1 / 4 of the amount used in Non-Patent Document 3. In addition, since this technology performs NMR measurement in one dimension, the measurement time can be reduced to about 1 / 10 of the experiment performed in Non-Patent Document 3.

[0093] It should be noted that the present invention is not limited to the embodiments described above, and various modifications are included. For example, this technology provides a program for performing each step of the method described above. This program may consist of one program or two or more subprograms.

[0094] Furthermore, for example, the embodiments described above are detailed explanations provided to clarify the present invention and are not necessarily limited to those comprising all the described configurations. It is also possible to replace parts of the configuration of one embodiment with those of another embodiment, and to add configurations from other embodiments to a given embodiment. Additionally, it is possible to add, delete, or replace parts of the configurations in each embodiment with those of other embodiments.

[0095] Furthermore, in diagrams showing the hardware configuration, only control lines and information lines deemed necessary for explanation are shown, and not all control lines and information lines are necessarily shown in the actual product. In reality, it can be assumed that almost all components are interconnected.

[0096] Furthermore, in the above embodiments, a specific example of a low-bonding affinity interaction was given for the case of the CH-π interaction, but the low-bonding affinity interaction formed between the first molecule and the second molecule is not limited to this example. For example, in hydrophobic interactions, dehydration can occur, which can lead to a large difference in chemical shift between the single state (before dehydration) and the complex (after dehydration). Those skilled in the art will understand, based on the description of the above embodiments and the general technical knowledge in this field, that this technology can be used to screen for the presence or absence of interactions (bonds) where a large difference in chemical shift between the single state and the complex can occur, such as CH-π interactions and hydrophobic interactions. Furthermore, the above-described embodiments disclose at least the configuration described in the claims. [Explanation of Symbols]

[0097] 100...Structural analysis system, 101...Magnet unit, 102...Spectrometer, 103...Control device, 51...CPU, 52...Memory, 53...Input / Output unit (IF), 54...Measurement unit, 55...Acquisition unit, 56...Evaluation unit, 57...Structural estimation unit, 104...Database, 105...User terminal

Claims

1. To obtain the first transverse relaxation rate in NMR measurement of the proton of the second molecule in a first sample containing a first molecule having an aromatic ring and a second molecule having a CH bond, To obtain the second transverse relaxation rate in NMR measurement of the proton of the second molecule in a second sample containing the second molecule in a standalone state, Based on the difference in transverse relaxation rates obtained by subtracting the second transverse relaxation rate from the first transverse relaxation rate, it is evaluated whether the first sample contains hydrogen atoms located near the aromatic ring within the first molecule. A method for structural analysis of a complex, including [the specified element].

2. Based on the difference between the first transverse relaxation rate and the second transverse relaxation rate, it is evaluated whether the first sample contains hydrogen atoms that form a CH-π interaction with the aromatic ring within the first molecule. The method according to claim 1.

3. Based on the difference between the first transverse relaxation rate and the second transverse relaxation rate, it is evaluated whether the first sample contains hydrogen atoms located within 6 Å of the aromatic ring within the first molecule. The method according to claim 1.

4. The second molecule is evaluated to determine whether it contains a hydrogen atom located within 5 Å, preferably within 4 Å, and more preferably within 3 Å, of the aromatic ring in the first molecule. The method according to claim 3.

5. If the difference is greater than or equal to a predetermined threshold, it is determined that the hydrogen atom is located near the aromatic ring in the first molecule. The method according to claim 1, including the method described in claim 1.

6. If the difference is greater than or equal to a predetermined threshold, it is determined that the number of hydrogen atoms in the second molecule are located near the aromatic ring in the first molecule equal to the number of protons for which the difference is greater than or equal to the predetermined threshold. The method according to claim 1, including the method described in claim 1.

7. The first lateral relaxation speed and the second lateral relaxation speed are, １ It is calculated based on the H NMR signal. If the difference is greater than or equal to a predetermined threshold, then the difference is greater than or equal to the predetermined threshold. １ It is determined that the hydrogen atom in the second molecule to which the H NMR signal is assigned is located near the aromatic ring in the first molecule. The method according to claim 1, including the method described in claim 1.

8. The second molecule in its standalone state １ Attribution information is obtained indicating whether part or all of the H NMR signal belongs to any of the second molecules. Based on the aforementioned attribution information, the hydrogen atoms in the second molecule located near the aromatic ring in the first molecule are identified. The method according to claim 7.

9. The predetermined threshold value is 0.05 S -1 or more, preferably 0.1 S -1 or more, more preferably 0.2 S -1 or more, more preferably 0.3 S -1 or more, more preferably 0.4 S -1 or more, more preferably 0.5 S -1 or more The method according to any one of claims 5 to 7.

10. The molecular weight of the second molecule is 150 to 3000. The method according to claim 1.

11. The molecular weight of the first molecule is 15,000 or more. The method according to claim 1.

12. The molecular weight of the first molecule is five times or more than the molecular weight of the second molecule. The method according to claim 1.

13. The aromatic ring in the first molecule is derived from the aromatic amino acid portion that constitutes the first molecule. The method according to claim 1.

14. The aromatic amino acid moiety is at least one selected from the group consisting of phenylalanine (molecular weight: 165.19), tryptophan (molecular weight: 204.23), histidine (molecular weight: 155.1546), and tyrosine (molecular weight: 181.19). The method according to claim 13.

15. To obtain the three-dimensional structural data of the first molecule, To obtain the three-dimensional structural data of the second molecule, Based on the positional information of the aromatic ring in the three-dimensional structural data of the first molecule and the positional information of hydrogen atoms in the three-dimensional structural data of the second molecule that have been identified as being located near the aromatic ring within the first molecule, the position and angle of the second molecule that is bonded to the first molecule are estimated. The method according to claim 1, including the method described in claim 1.

16. In the first sample, the molar concentration of the second molecule is three times or more, preferably ten times or more, the molar concentration of the first molecule. The method according to claim 1.

17. To obtain the first transverse relaxation velocity １ For obtaining the H NMR signal and the second transverse relaxation velocity １ At least one of the H NMR signals is acquired based on the CPMG method. The method according to claim 7.

18. An acquisition unit that acquires a first transverse relaxation rate in an NMR measurement of the proton of a second molecule in a first sample comprising a first molecule having an aromatic ring and a second molecule having a CH bond, and a second transverse relaxation rate in an NMR measurement of the proton of a second molecule in a second sample comprising the second molecule in a standalone state. An evaluation unit that evaluates whether the first sample contains hydrogen atoms located near the aromatic ring within the first molecule, based on the difference between the first transverse relaxation rate and the second transverse relaxation rate, A structural analysis device for composites, equipped with the necessary components.

19. The three-dimensional structural data of the first molecule and the three-dimensional structural data of the second molecule are obtained. A structural estimation unit estimates the position and angle of the second molecule that is bonded to the first molecule, based on the position information of the aromatic ring in the three-dimensional structural data of the first molecule and the position information of hydrogen atoms in the three-dimensional structural data of the second molecule that have been identified as being located near the aromatic ring in the first molecule. The apparatus according to claim 18, further comprising:

20. A program for causing a computer to perform each step of the method for analyzing a composite structure as described in claim 1.

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