JP2003279566A - Method for screening agent capable of bonding to receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel screening method which can solve problems of conventional screening methods such as a method of SAR by NMR. <P>SOLUTION: The method for screening an agent capable of optimally bonding to a receptor by using the one molecule fluorescence analysis is provided with a process in which the agent capable of bonding to the receptor is searched, and a process is which the agent capable of bonding to the receptor obtained in the above process is optimized in structure. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for screening a substance capable of binding to a receptor. In particular, the method of the present invention is useful for structure-activity relationship analysis for efficiently discovering a ligand and designing a drug discovery target for a novel drug discovery target.

[0002]

BACKGROUND OF THE INVENTION (Background in drug discovery screening) Drug discovery is the discovery and creation of molecules having drug efficacy or applicable to the treatment of diseases. In the process,
This is a difficult and time-consuming and costly work that must be screened after taking into consideration the efficacy, bioavailability, biodistribution, metabolic rate, excretion rate, side effects and toxicity.

Drug discovery research is roughly divided into two research cycles (see FIG. 1; Greer J, Erickson JW, Baldwin J.
J, Varney MD., J Med Chem 1994 Apr 15; 37 (8): 103
5-54: Application of the three-dimensional structur
es of protein target molecules in structure-based
Refer to drug design.). The first cycle (shown in FIG. 1) is a “cycle in which structural modification of lead compounds and evaluation of drug efficacy are repeated while taking into account quantitative structure-activity relationships, relying on the experience and intuition of synthetic researchers”. This cycle was mainstream until the 1980s. In this cycle, due to the progress of high-throughput screening technology by combinatorial chemistry and robotization in recent years, the time required for synthesis and drug efficacy evaluation has been significantly shortened, and it has evolved into synthesis and screening of hundreds of thousands of compounds. However, whether or not a hit compound having sufficient activity can be obtained depends largely on the screening system and the compound library to be screened, and thus, in some cases, a hit compound could not be obtained.

On the other hand, as a result of the gradual increase in the results of protein three-dimensional structure analysis since the latter half of the 1980s, drug design was experimentally conducted based on the three-dimensional structure and interaction analysis of the target protein / drug complex. -based dr
ug design (SBDD) has emerged. Further, a method of constructing a three-dimensional structure model of a target protein by homology modeling and designing a compound capable of interacting by docking on a computer has also been generally used. These are shown in FIG.
-3 drug discovery cycles are formed.

Nowadays, with the development of structural biology, many three-dimensional structures of biopolymers have been clarified by X-ray crystallography and nuclear magnetic resonance (NMR). On the other hand, with the progress of the Structural Genomics Science Project as part of the Post Genome Project,
From 2005 to 2010, the new three-dimensional structures of about 10,000 kinds of proteins will be analyzed and will be published on the database. Furthermore, if there is a day when typical structures of almost all protein families will be registered in the database, it is possible to construct a 3D structure by homology modeling from the 3D structures of related proteins in the database for any drug target. It will be possible. It is highly possible that structure-based drug design (SBDD) will take root in Japan in the near future based on the research results of the Structural Genomics Science Project.

However, what is important in the drug discovery cycle of FIG. 1 is that all the cycles do not start unless the lead compound or its candidate compound is identified. Even if a drug target protein is selected from genomic information and its three-dimensional structure is immediately available, it is still difficult to create a lead compound from only the three-dimensional structure of the protein, and it has a high affinity for that protein.・ The situation where we have to search for compounds that bind with high selectivity has not changed.

On the other hand, as a method for analyzing a three-dimensional structure, 19
In 1996, the group of SW Fesik, "lead compound creation method combining compound screening by NMR and linked fragment approach", so-called SAR by NMR
(Structure-activity relationship by nuclear magne
tic resonance) (reference: Shuker SB, Ha
jduk PJ, Meadows RP, Fesik SW., Science, 1996 Nov
29, 274 (5292), 1531-4, Discovering high-affinity
ligands for proteins: SAR by NMR). NMR is recognized as a method for analyzing the three-dimensional structure of proteins, and 19
In the early 90's, it was introduced by pharmaceutical companies for the purpose of SBDD. Prior to that, due to its low sensitivity and time-consuming experimentation, screening with NMR had not been considered, and thus attracted great attention.

(Drug screening by NMR) NM
R is now widely applied as a three-dimensional structural analysis method for biopolymers such as proteins and nucleic acids together with X-ray crystallography. However, the application of NMR is limited to molecules having a molecular weight of 20,000 or less. Generally, molecules of 20,000 or less are standard sizes as a unit of a functional domain to which a drug binds. However, it takes several days to one month to analyze a three-dimensional structure by NMR, and it is practically impossible to analyze each three-dimensional structure for the purpose of compound screening. Even if the lead compound has already been found, it is more efficient to synthesize and assay the candidate compound using combinatorial chemistry rather than spending time on the three-dimensional structure analysis and drug design from there.

The NMR analysis and its features will be described below. The advantage of NMR is that
The NMR signal sensitively reflects local environmental changes of the molecule (drug binding, structural changes, changes in motility, etc.). For example, when a protein (P) and a ligand (L) bind to each other to form a complex (PL), the NMR spectrum of the complex (PL) has P, L
Is different from when each exists alone.
Using the change in the spectrum as an index, a protein-drug binding assay can be performed. The features of NMR when used for random screening are listed below.

NMR has lower sensitivity than spectroscopy such as UV, CD and fluorescence, and requires a protein at a concentration of 0.2 mM or more. Further, the sample for one measurement requires 250 to 300 μL. Generally, for a protein sample of about 0.3 mM, it takes several minutes for one-dimensional NMR and about 15 minutes for two-dimensional NMR. ¹⁵ N using a protein uniformly labeled with ¹⁵ N-
^{Using 1} HHSQC spectrum, N
Only MR signals can be observed. If the NMR signal of the protein is assigned, the drug binding site can be identified from the signal changed when the drug is added.

Regarding the low sensitivity of measurement of features,
The disadvantage is that both the protein and the ligand compound need to be dissolved in water at high concentrations. In terms of characteristics, the speed of the assay is limited, and it is not suitable for high throughput. 1 to increase processing speed
It is practically difficult to prepare many high magnetic field NMR apparatuses that cost hundreds of millions of yen.

[0012] On the other hand, the characteristic is an advantage that other spectroscopy does not have, and even if a drug is added at a high concentration (excess), the protein spectrum can be measured without being disturbed by the signal of the drug. Furthermore, the advantage of the characteristics is that the sample concentration is high, so that the affinity is low (even if the dissociation constant is about 0.1 to 1 mM).
It is possible to find compounds that bind site-specifically to proteins. In particular, compounds that show low affinity for the target protein are not considered hit compounds in conventional assay systems, but such compounds lead to site-specific binding to proteins. It is an important candidate compound that can be a partial structure of a compound. Compared to other assays that use chemical reactions and biological responses,
The advantage of NMR is that hit compounds can be screened with binding site information and direct protein-drug interactions can be detected.

SAR by NMR is the structure-activity relations obtained by NMR.
It is an abbreviation for p), and is a method that includes search for lead compounds and structure optimization. This method involves "screening of low molecular weight compounds (fragments) that bind to a target protein using NMR" and "improvement of affinity by linking the compounds obtained by screening with a covalent bond via a linker (linked fragment approach) ”.

FIG. 2 shows the outline of SAR by NMR. The steps from to in FIG. 2 will be described.

A compound that directly interacts with a specific site (binding site a) of a target protein from a compound library using a change in NMR chemical shift when a ligand is added to a ¹⁵ N-labeled target protein solution as an index (hit compound ) Is screened. The structure of the hit compound is modified to find the compound that binds most strongly to the binding site a (structure-activity relationship). In the presence of the compound obtained in step b
Hit compounds that bind to are screened as in step. In the same manner as in the step of (2), the structure of the hit compound is optimized for the binding site b (structure-activity relationship). By covalently linking the compounds that bind to the two sites a and b, the affinity for the target protein is improved (linked fragment approach).

In this method, the compound corresponds to an average molecular weight of up to more than 200, and it cannot be measured unless care is taken so that the molecular weight does not become too large in the step.

By the above method utilizing NMR, FK
There is a report searching for an inhibitor of 506 binding protein (FKBP) (Shuker SB, Hajduk PJ, Meadows RP, Fesi
k SW., Science, 1996 Nov 29, 274 (5292), 1531-4, D
iscovering high-affinity ligands for proteins: SAR
by NMR). With this method, FK uniformly labeled with ¹⁵ N
A library screen of about 1000 compounds was performed using BP, and it was detected that two kinds of compounds had high affinity for FKBP when they bound to FKBP.

The screening of compounds (fragments) by the above-mentioned NMR spectroscopy has the following problems.

(1) The high magnetic field NMR, which is a measuring instrument required for drug discovery screening, is extremely expensive at 100 to 200 million yen per unit, and requires a special sample tube and an automatic sample changer. (2) Since the operation of the equipment requires a great deal of expertise, N
Both MR technicians and even biochemists are needed. (3) The sample requires a large amount (200 mg or more) of ¹⁵ N-labeled target protein, and also requires a high sample concentration of 0.2 mM or more. Also,
A high-purity sample is required for NMR measurement, and a crude product containing the target protein cannot be used as a sample. Therefore, sample preparation is complicated and time-consuming. (4) The measurable molecular weight is limited to 20,000, and 2
It cannot be used for proteins of 0 kDa or more. NMR
Since it is necessary to examine each protein domain, it is not suitable for screening a drug having a binding site across multiple domains. (5) It is unsuitable for measurement of samples with complicated structures such as lipids and glycoproteins. (6) The target protein or compound cannot be used unless its structure is known. Therefore, when the side chain of a compound is randomly modified, the compound cannot be immediately used for measurement. (7) Although it is possible to detect whether or not the test compound binds to the target protein, it is not possible to examine the strength of the binding force. (8) Since NMR measurement takes time, it is not suitable for high throughput screening.

[0020]

In view of the above circumstances, it is an object of the present invention to provide a novel screening method capable of solving all the problems of conventional screening methods such as SAR by NMR.

[0021]

The present inventors have found a new method for screening a substance capable of specifically binding to a receptor simply and quickly by using a non-radioactive substance as a detection marker. The invention was completed. It should be noted that the method of the present invention solves all the problems of conventional screening methods such as SAR by NMR.

That is, the present invention provides the means described below.

(1) A method for screening a substance capable of optimally binding to a receptor by single molecule fluorescence analysis, which comprises a step of searching for a substance capable of binding to the receptor and the receptor obtained by the above step. Performing the structural optimization of the bondable substance.

(2) A method for screening a substance capable of binding to a receptor having a first binding site and a second binding site by single molecule fluorescence analysis, which is a substance capable of binding to the first binding site And a substance capable of binding to the second binding site under the condition that the substance capable of binding to the first binding site obtained by the above process binds to the receptor. And a step of: (3) A method for screening a substance capable of optimally binding to a receptor having a first binding site and a second binding site by single molecule fluorescence analysis, wherein a substance capable of binding to the first binding site is selected. A step of searching, a step of optimizing the structure of a substance capable of binding to the first binding site obtained by the step, and a substance capable of optimally binding to the first binding site obtained by the step The step of searching for a substance capable of binding to the second binding site under the condition of binding to the receptor, and the structure of the substance capable of binding to the second binding site obtained by the step And a step of performing optimization. (4) The method according to (3), wherein the length of the linker for connecting the substance capable of optimally binding to the first binding site and the substance capable of optimally binding to the second binding site Now, the method further comprising the step of preparing a substance capable of optimally binding to the receptor by screening for the structure.

(5) Screening a substance capable of binding to a receptor having n (n is an integer of 2 or more) binding sites from the first binding site to the nth binding site by single molecule fluorescence analysis And a step of searching for a substance capable of binding to the first binding site, and capable of binding to (X-1) binding sites from the first binding site to the (X-1) th binding site. A step of sequentially searching (N-1) times from X = 2 to X = n under the condition that each substance binds to the receptor. And a method comprising: (6) From the first binding site to the nth molecule by single molecule fluorescence analysis
Is a method for screening a substance capable of optimally binding to a receptor having n (n is an integer of 2 or more) binding sites up to the binding site of From the first binding site to the second step of optimizing the structure of the substance capable of binding to the first binding site obtained in the first step. )
Up to the binding site of (X-1), each of the substances capable of binding to the binding site is optimally bound to the receptor, and a substance capable of binding to the Xth binding site is searched for. And a fourth step of optimizing the structure of a substance capable of binding to the X-th binding site obtained by the third step, wherein the third step and the fourth step are: X = 2 to X =
A method characterized by being repeated (n-1) times in sequence up to n. (7) The method according to (6), wherein n substances are linked from a substance capable of optimally binding to the first binding site to a substance capable of optimally binding to the nth binding site. The method further comprising the step of preparing a substance capable of optimally binding to the receptor by screening for the length and structure of the linker to be used.

(8) The method according to any one of (1) to (7), wherein the substance capable of binding to the receptor is a target drug in drug discovery. (9) The substance capable of binding to the receptor is labeled with a photodetectable substance (1) to
The method according to any one of (8).

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION <Single Molecule Fluorescence Analysis Technique> First, the analysis technique and single molecule fluorescence analysis used in the screening method of the present invention will be described.

The single-molecule fluorescence analysis is a technique for measuring fluctuation motions of fluorescent molecules that move in and out of a minute confocal region, and analyzing the data obtained by this measurement by a function.
This technology includes (1) Fluorescence C
orrelation Spectroscopy), (2) Fluorescence Intensity DistributionAna
(3) and (3) Fluorescence Intensity Multiple Dist
ribution Analysis) is included. Each will be described below.

(1) Fluorescence correlation spectroscopy (hereinafter also referred to as FCS) measures the fluctuation motion of a fluorescence-labeled target molecule in a medium to obtain an autocorrelation function.
ion) is a technique for accurately measuring the micromotion of individual target molecules (reference: D. Magde and
E. Elson, “Fluorescence correlation spectroscopy.
II. An experimental realization ”, Biopolymers 19
74 13 (1) 29-61).

In FCS, the Brownian motion of fluorescent molecules in a solution is captured in a minute region by a laser confocal microscope to analyze the diffusion time from fluctuations in fluorescence intensity and to measure physical quantities (number and size of molecules). It is executed by FCS that captures molecular fluctuations in such a small area
The analysis by is effective for highly sensitive and specific detection of intermolecular interactions.

The principle of detection by FCS will be described in more detail. In FCS, a fluorescence signal generated from a microscopic field area in a sample is detected and quantified by a microscope. At this time, the fluorescently labeled target molecule in the medium is always in motion (Brownian motion). Therefore, the detected fluorescence intensity changes depending on the frequency with which the target molecule enters this microscopic visual field region and the time for which the target molecule remains in the microscopic visual field region.

For example, if the apparent dimerization of molecules occurs and the apparent molecular weight increases, the movement of the target molecule slows down and the apparent number of molecules decreases. As a result, the frequency of entering the microscopic visual field region is reduced, and the observed fluorescence intensity is changed. By monitoring such changes in fluorescence intensity, changes in the apparent molecular weight of the target molecule can be tracked.

(2) Fluorescence intensity distribution analysis (hereinafter also referred to as FIDA) is described in the reference P Kask, et al; PNAS 23, 96, 13756-.
13761, 1999, WO98 / 16814. FIDA
Irradiates the sample with a laser and emits the excitation light of fluorescent molecules emitted from the sample to the APD (high-sensitivity photodetector).
It is a technique for performing double Poisson distribution function analysis and statistical processing analysis for the photon count obtained by measuring the measured fluorescence signal in a very short time of 40 microseconds. FIDA calculates the fluorescence intensity (brightness; qn) and the number of fluorescent molecules (cn) per molecule (see FIG. 3). Even if there are multiple types of molecules, they can be identified by distribution analysis, and the value obtained by multiplying the brightness and the number of molecules for each type of molecule can be calculated as the total fluorescence amount. FIG. 3 shows an example of Poisson distribution function analysis of fluctuations in the fluorescence intensity of fluorescent molecules that enter and leave a small confocal region.

(3) Fluorescence intensity multi-distribution analysis (hereinafter referred to as FIMD
(Also referred to as A) is for performing FCS analysis and FIDA at the same time. For details, refer to the document K Palo, Biophysical J
ournal, 79, 2858-2866, 2000). F
According to IMDA, translational diffusion time of fluorescent molecule, number of molecules,
And the data regarding the fluorescence intensity (brightness) per molecule can be acquired at the same time. Therefore, not only the size of the molecule can be determined, but also about 5 times change in the size of the molecule, which cannot be identified by FCS, can be identified by the difference in brightness.

<Screening Method of the Present Invention> The screening method of the present invention can be carried out using the above-mentioned single-molecule fluorescence analysis technique. Hereinafter, the method of the present invention will be described in detail.

The screening method of the present invention is a method for screening a substance capable of optimally binding to a receptor by single molecule fluorescence analysis, and is obtained by the step of searching for a substance capable of binding to a receptor and the above-mentioned steps. And optimizing the structure of a substance capable of binding to the receptor.

(Receptor) The receptor used in the method of the present invention may be any receptor, which accepts a ligand in vivo and causes various responsive reactions in vivo by its receptor signal. There is no particular limitation as long as it exists. In particular, a receptor in which various response reactions in vivo due to the receptor signal are associated with a disease is preferably used. The size of the receptor used in the method of the present invention is not particularly limited, and can be various sizes, for example, from 0.4 kDa to 800 kDa. Examples of the receptor used in the method of the present invention include GPCR (G protein-coupled receptor)
And so on.

The receptor may be prepared in any manner, for example, a gene expressed in vitro by a known genetic engineering technique using a gene encoding the receptor may be used. Alternatively, it may be a crudely purified product obtained by collecting a fraction containing the receptor from the living body.

(Step of searching for substance capable of binding to receptor) In the present invention, the substance capable of binding to the receptor is any substance which is suspected to bind to the above-mentioned receptor and cause a response reaction in the living body. (Hereinafter also referred to as a candidate substance) is searched. In the present invention, candidate substances include both organic compounds and inorganic compounds, examples of which include chemical substances such as proteins, glycoproteins, lipids, amino acids and other molecules that make up the body, hormones, autacoids, ions, metals, etc. Is mentioned.

In the present invention, at least one of the candidate substance and the receptor needs to be labeled for single molecule fluorescence analysis. Hereinafter, the cases of (A) to (C) will be described as representative patterns of signs, but the present invention is not limited thereto.

(A) When labeling a candidate substance In this case, since the free candidate substance and the candidate substance bound to the receptor obviously have different molecular sizes, they can be distinguished by single molecule fluorescence analysis. (B) When labeling a receptor The receptor can be labeled if it causes a size change of the receptor molecule such as dimerization of the receptor molecule after the receptor receives the ligand. That is, the fact that the receptor has received the candidate substance can be identified by the single molecule fluorescence analysis based on the size change of the receptor molecule. (C) When both the candidate substance and the receptor are labeled with the same labeling substance When the candidate substance binds to the receptor, the fluorescence intensity per receptor molecule doubles. Can be identified by analysis.

As the marker substance for labeling, any substance can be used so long as it emits a signal that can be optically traced, that is, a substance capable of photodetection. Various dyes that generate stable signals that can be quantified are preferable,
In particular, a fluorescent dye that can individually measure the presence of minute substances is preferable. Preferably, any fluorescent substance that emits detectable fluorescence can be used. For example, fluorescein isothiocyanate (FITC), rhodamine, TAMRA, C
y3 (Amersham Pharmacia), Cy5 (Amersham
It is possible to use various fluorescent dyes such as Pharmacia. Fluorescent labeling can be performed by a known method.

The step of searching for a substance capable of binding to the receptor is carried out by placing the receptor and the candidate substance in a predetermined solution. The predetermined solution may be a solution capable of binding the receptor and the original ligand of the receptor. For example, physiological saline or phosphate buffer can be used.

By keeping the receptor and the candidate substance under the predetermined condition in this predetermined solution, only the substance capable of binding to the receptor among the candidate substances causes the binding reaction. Here, the predetermined conditions (temperature, pH, reaction time, etc.) can be appropriately set depending on the type of the receptor.

The amount of the candidate substance added to the reaction solution is preferably such that the final concentration is about 0.01 to 100 nM, and more preferably about 0.1 to 50 nM. The amount of the receptor to be added is 0.01 nM to 10 μM in the final concentration.
The amount is about 0.1 to 50, more preferably 0.1 to 50.
About nM is preferable.

As an example of the reaction, a solution containing a receptor (FK506 binding protein) in an amount of 10 nmol / L in a physiological buffer solution and 5-T as described in Examples below.
The candidate substance labeled with AMRA is added to a physiological buffer solution at 1 to
It can be carried out by mixing 50 μL each with a solution containing 100 nmol / L and incubating at room temperature for 15 to 30 minutes.

In order to incubate, a reaction solution containing a set of reaction components in a floating state in the above-mentioned predetermined solution is added to an appropriate liquid holding means such as a test tube, a well, a cuvette, a groove, a tube, a flat plate, and a porous plate. It can be held on the body or the like. Here, the shape, material, size and the like of the liquid holding means are preferably selected so that some or all of various detection steps such as dispensing, stirring, incubating, measuring, and transporting can be performed quickly. For example, the measurement by single molecule fluorescence analysis uses a very small area of an optical focus level as a measurement place, and thus can be a very small liquid containing means. In particular,
In the detection by optical measurement, the liquid holding means preferably has an opening through which the measurement beam enters and / or exits, so that the measurement light beam is directly related to the reaction component as much as possible.

In the solution after the above reaction, the abundance ratio of the candidate substance bound to the receptor and the candidate substance released in the reaction solution is measured by single molecule fluorescence analysis based on the difference in their molecular weights. can do. Thereby, the ease of binding (affinity) of the candidate substance to the receptor can be obtained as a dissociation constant.

When a substance capable of binding to the receptor is found from the candidate substances, the structure of the substance is optimized. When the screening method of the present invention is applied to drug discovery screening, all substances capable of binding to the receptor can be referred to as “hit compounds”. Of the “hit compounds”, the compound that has strong binding to the receptor and is transferred to the subsequent structural optimization step is the “lead compound”.
Can be said.

Hereinafter, the substance from which structure optimization is performed for convenience will be referred to as a "lead compound", but it goes without saying that the screening method of the present invention is not limited to the application of drug discovery screening.

Structural optimization refers to modification and alteration of the side chain of a lead compound to obtain compounds having various structures (for example, 5 to 5).
20), and the ability of each of these various compounds to bind to the receptor is investigated, and the optimum one is selected. In the present invention, optimization is an operation of selecting a compound having the highest binding force from various compounds prepared by modification / alteration.

Examples of modification / modification of the side chain include modification of the amide group contained in the compound to a carboxyl group or a hydroxyl group. An example in which the side chain of the compound is actually modified for structural optimization is described in the drawing (FIG. 6) illustrating the example. However, the present invention is not limited to this example, and any side chain can be modified / modified, and those skilled in the art can appropriately modify / modify the side chain.

The ability of the compound to bind to the receptor can be determined by single molecule fluorescence analysis based on the change in molecular weight, the change in fluorescence intensity per receptor molecule, etc., as described above.

(Preferred Embodiment) As a preferred embodiment, an example in which the screening method of the present invention is applied to drug discovery screening will be described below. An outline of the preferred embodiment is shown in FIG. However, it goes without saying that the present invention is not limited to the following embodiments.

That is, a preferred embodiment of the present invention is a method for screening a substance capable of optimally binding to a receptor having a first binding site and a second binding site by single molecule fluorescence analysis, A step of searching for a substance capable of binding to the binding site of, a step of optimizing the structure of the substance capable of binding to the first binding site obtained by the step, and the first step obtained by the step of Under the condition that a substance capable of optimally binding to the binding site is bound to the receptor,
A step of searching for a substance capable of binding to the second binding site; a step of optimizing the structure of the substance capable of binding to the second binding site obtained by the step; A substance capable of optimally binding to the receptor is prepared by screening for the length and structure of a linker for linking the substance capable of binding to the second binding site and the substance capable of optimally binding to the second binding site. It is characterized by comprising a step of

The above-mentioned steps (1) to (4) correspond to (1) to (4) in FIG. The steps will be described below in order.

First, the receptor used here is a site a to which a substance capable of binding to the receptor (also called substance a) is bound.
In addition to the binding site a, it also has a binding site b to which another substance (also referred to as a substance b) binds. In drug discovery screening, the binding site a and the binding site b are generally preferably located near each other. Here, “vicinity” means that the binding site a and the binding site b are sterically arranged so that they can be sterically linked by a linker.

It is well known to those skilled in the art that the screening of substances capable of binding to each of the two sites in this way is an effective method in drug discovery screening when developing highly effective drugs.

In the step (1), a substance (substance a) capable of binding to one of the two binding sites (binding site a in FIG. 4) is searched for. See the above description for this step.

In the step (1), the structure of the substance capable of binding to the binding site (a) obtained in the above step is optimized. See also the above description for this step.

In the step (1), a substance (substance (substance) that can optimally bind to the binding site (a) obtained in the above step is bound to another binding site (b) under the condition that it binds to the receptor. Search b). The step can be performed in the same manner as the step.

Here, it is preferable that the substance a and the substance b are discriminatively labeled with two kinds of fluorescent substances each having a different excitation wavelength, as shown in FIG. Accordingly, the binding of the substance a to the receptor and the binding of the substance b to the receptor can be identified and recognized. Any labeling substance may be used as long as it emits a signal that can be optically tracked, and is as described above.

Further, "under the condition that the substance capable of optimally binding to the binding site a is bound to the receptor" means that the substance capable of optimally binding to the binding site a is in the same solution as the receptor. It means the condition that the substances bound to the receptor and the free substances that do not bind are allowed to exist in equilibrium.

In the step, the structure of the substance capable of binding to the binding site b obtained in the above step is optimized. The step can be performed in the same manner as the step.

Regarding the length and structure of the linker for connecting the substance capable of optimally binding to the binding site a (substance A) and the substance capable of optimally binding to the binding site b (substance B) in the step of To screen. Thereby, a substance capable of optimally binding to the receptor can be prepared.

The linker is not particularly limited as long as it can connect the substance A and the substance B. For example, as described in Examples below, those in which methylene groups (—CH ₂ — groups) are connected in various numbers can be used.

<When Receptor has Two or More Binding Sites> In the case where the receptor molecule has two or more binding sites, it is possible to screen a substance capable of binding to the receptor molecule in the same manner. is there. That is, the method for screening a substance capable of binding to a receptor having two or more binding sites is preferably as described below.

That is, a substance capable of optimally binding to a receptor having n (n represents an integer of 2 or more) binding sites from the first binding site to the nth binding site is determined by single molecule fluorescence analysis. A screening method, which comprises a first step of searching for a substance capable of binding to a first binding site, and structural optimization of a substance capable of binding to the first binding site, obtained by said first step. And a substance capable of binding to the (X-1) th binding site from the first binding site to the (X-1) th binding site is optimally bound to the receptor, respectively. Under the conditions that the third step of searching for a substance capable of binding to the X-th binding site and the structure optimization of the substance capable of binding to the X-th binding site obtained by the third step. Four steps, wherein the third step and the fourth step are X
= 2 to X = n sequentially (n-1) times, further, from the substance capable of optimally binding to the first binding site to the substance capable of optimally binding to the nth binding site It is characterized by comprising a fifth step of preparing a substance capable of optimally binding to the receptor by screening up to n substances for the length and structure of a linker for connecting each of them.

The screening of a substance capable of binding to a receptor having two or more binding sites is similar to the method of screening a substance capable of binding to a receptor having two binding sites, and the description is appropriately referred to. I want to be done. Generally, in the drug discovery scene, a receptor having 2 to 5 binding sites is often assumed.

<Device for Performing Single Molecule Fluorescence Analysis> An example of a device for performing single molecule fluorescence analysis (hereinafter, also referred to as fluorescence correlation analyzer) will be described with reference to FIG.

As shown in FIG. 5, the fluorescence correlation analyzer is
Laser light source 1 and light intensity adjusting means (here, ND filter) for reducing the intensity of the light beam from the laser light source 1.
2, an optical attenuation selection device (here, an ND filter changer) 3 for setting an appropriate light intensity adjusting means 2, and an optical system for condensing a light beam from the laser light source 1 on the sample to form a confocal region. 4 and 5, a stage 6 on which a sample containing fluorescent molecules is placed, optical systems 7 to 11 for collecting fluorescence from the sample, and a photodetector 1 for detecting the collected fluorescence.
2 and fluorescence intensity recording means 13 for recording the change in fluorescence intensity
It is equipped with. Thus, the fluorescence correlation analyzer uses a confocal laser microscope.

In FIG. 5, the laser emitted from the laser light source 1 may be any of an argon ion laser, a helium-neon laser, a krypton, a helium-cadmium and the like. In FIG. 5, the optical systems 4 and 5 that focus the light beam from the laser light source on the sample to form the confocal region are
Specifically, it means the dichroic mirror 4 and the objective lens 5. The light beam from the laser light source 1 is attenuated in accordance with the attenuation degree of the fluorescence intensity adjusting means (here, the ND filter) 2 along the path shown by the arrow in FIG. 5, and then is incident on the dichroic mirror 4. The light is refracted by 90 degrees in the direction of the stage, passes through the objective lens 5, and is irradiated onto the sample on the stage 6. In this way, the light beam is focused on the sample at one minute point to form a confocal region.

In FIG. 5, the optical systems 7 to 11 which collect the fluorescence emitted from the fluorescent molecules in the confocal region are specifically the filter 7, the tube lens 8, the reflecting mirror 9, the pinhole 10 and the lens 11. Means Fluorescence emitted from the fluorescent molecule is first transmitted through the dichroic mirror 4 in the light traveling direction in a path as shown by an arrow in FIG. 5, passes through the filter 7, the tube lens 8, and is refracted by the reflecting mirror 9, After forming an image on the pinhole 10, the light passes through the lens 11 and is focused on the photodetector 12.

The photodetector (here, avalanche photodiode) 12 for detecting the collected fluorescence converts the received light signal into an electric signal and sends it to the fluorescence intensity recording means (here, computer) 13.

The fluorescence intensity recording means 13 for recording the change in fluorescence intensity records and analyzes the transmitted fluorescence intensity data. Specifically, the autocorrelation function is set by analyzing the fluorescence intensity data. The increase in molecular weight due to the binding of the fluorescent molecule to the receptor and the decrease in the number of free fluorescent molecules are
It can be detected by a change in the autocorrelation function.

The apparatus for performing single molecule fluorescence analysis (fluorescence correlation analysis apparatus) is not limited to the example shown in FIG. For example, as shown in FIG. 4, the substance a that binds to the binding site a and the substance b that binds to the binding site b can be labeled with two kinds of fluorescent substances each having a different excitation wavelength so as to be bound to each binding site When screening substances, the fluorescence correlation analyzer has two types of laser light sources for exciting each fluorescent substance, and further two types of light sources for detecting light emitted from each fluorescent substance. It is necessary to have a photodetector. The photodetector is A
In addition to the PD (avalanche photodiode), a device including a photomultiplier tube may be used.

<Regarding the Effect of the Present Invention> As described above, the screening method using the single-molecule fluorescence analysis method of the present invention is compared with the conventional structure-activity relationship (SAR by NMR) using NMR as an analysis method. , Has the following advantages.

(1) Although the NMR apparatus is very expensive at 200 to 300 million yen, the fluorescence correlation analyzer used in the present invention is low in price (about tens of millions) and the measurement cost is also high. Low.

(2) The operation method and NMR require not only the synthesis and handling of radioactive compounds but also the knowledge of NMR. However, the fluorescence correlation analyzer does not require special specialized ability and the operation is simple. It has become.

(3) The sample solution used in the method of the present invention is sufficient to have several tens of microliters. In addition, there is an advantage that the sample in the sample solution can be assayed with high sensitivity even if it has a low concentration.

(4) In addition to the acceptor molecules that can be used in the method of the present invention having a molecular weight of up to 20 kDa that can be measured by the NMR method, large molecules of 100 kDa or more can also be used. Further, the measurement of NMR requires that the receptor has a high purity, but in the present invention, it is not necessary to purify the receptor molecule used. Further, in NMR, since the structure analysis of a large molecule becomes complicated, it was necessary to prepare several kinds of radiolabels for each atom, but in a fluorescence correlation analyzer, by labeling with a fluorescent label, It can be easily analyzed that the body has received the substance. Further, in the case of a molecule having a size of 50 kDa or more, such as an acceptor molecule, in NMR, it was necessary to separate each domain by a genetic engineering method. No DNA recombination step is required. Therefore, the method of the present invention exerts a particularly excellent effect in screening for a drug having a binding site across a plurality of domains.

(5) Even a sample having a complex structure such as lipid or glycoprotein, which was unsuitable for NMR measurement, can be measured by the present invention using the single molecule fluorescence analysis technique.

(6) It could not be used for measurement unless the structures of the receptor and the substance that binds to it were known by NMR, but even if the structures are not known, they can be used in the screening method of the present invention. is there. Thus, in the screening method of the present invention, a substance in which the side chain of the compound is randomly modified can be used as a candidate substance.

(7) Although it was possible to detect whether or not the receptor binds to the test substance by NMR, it was not possible to examine the strength of the binding force, but in the present invention, single molecule fluorescence was detected. By applying the analytical technique, the abundance ratio of the test substance bound to the receptor and the test substance in the free state can be determined. Thereby, the binding force (affinity) between each substance and the receptor can be determined.

(8) In NMR, a very long measurement time (one month in some cases) was required, but in the present invention, by applying the single molecule fluorescence analysis technique, a short measurement time can be obtained. It is possible. Generally, a few seconds to several tens of seconds are sufficient for measurement. This makes the method of the present invention suitable for high throughput screening.

[0086]

Examples FK506 binding protein (FK506 bin
For the search for inhibitors of ding protein (FKBP), screening with a fluorescence correlation analyzer was performed. The outline is shown in FIG.

FKBP is a potent immunosuppressant FK
When bound to 506 (tacrolimus), it blocks activation of T cells by inhibiting calcineurin, which is a serine / threonine dephosphorylating enzyme.

As a target protein, FKBP was used and adjusted to a concentration of 10 nmol / L in a physiological buffer solution. The plurality of compound libraries (also referred to as the first compound library) are 5-TAMRA (5-Carboxytet
Ramethylrhodamine) was used for fluorescent labeling and adjusted to 3 concentrations (1, 10, 100 nmol / L) (hereinafter,
The fluorescently labeled compound is referred to as a first fluorescently labeled compound). Target protein FKBP and 3 concentrations (1,
The first fluorescently labeled compound (10,100 nmol / L, etc.) was mixed in the same amount of 50 μL. 15 at room temperature
After incubating for -30 minutes, the cells were placed in a 96-well glass bottom microplate and counted by a fluorescence correlation analyzer.

The laser is a helium-neon laser 543n.
m was used. The measurement time was 10 seconds per sample, and continuous measurement was performed 5 times, and the average value was used as data. As measurement parameters, translational diffusion time and molecular concentration were calculated, and the affinity of each compound was examined by Kd value (dissociation constant).

By the translational diffusion time, the first fluorescence-labeled compound alone and the target protein (FKBP) that interacted with the compound were discriminated and discriminated as two kinds of molecules. The first fluorescent labeling compound has a molecular weight of 70
0-1700, the interacted target protein (FKBP), has a molecular weight of about 30 kDa. Therefore, the translational diffusion time by the single molecule fluorescence correlation analyzer is
It was about 60 μs for the first fluorescent labeling compound and about 240 μs for the interacting target protein (FKBP). Due to the difference in the translational diffusion time, it was possible to distinguish among the same sample and calculate the respective molecular concentrations.

Some of the plurality of first fluorescently labeled compounds showed affinity with the target protein (FKBP). The compound showing this affinity was further rescreened, and the trimethoxyphenylpipecolic acid derivative (Compound 2 shown in FIG. 6) had a sufficiently high affinity (Kd = 2).
μM).

Next, compound 2 and target protein F
A mixed solution of KBP was prepared. In this mixed solution, the compound 2 interacts with FKBP, whereby the one bound to FKBP and the one bound to FKBP in a free state are mixed in an equilibrium state. Using such a mixed solution, a compound that binds in the vicinity of the compound 2 that interacted with FKBP was screened from several compound groups (also referred to as a second compound library).

The second compound library was labeled with Cy5 (Amersham) as a fluorescent substance (hereinafter, the fluorescently labeled compound is referred to as a second fluorescently labeled compound). In the fluorescence correlation analyzer, helium neon laser 633nm
Was irradiated and measured. The second fluorescent labeling compound uses an excitation wavelength different from that of the above-mentioned first fluorescent labeling compound upon detection. Therefore, by irradiating with a wavelength that excites only the second fluorescent labeling compound, only the sample in which the two types of fluorescent labeling compounds interact can be screened.

The translational diffusion time by the fluorescence correlation analyzer is
It was about 60 μsec for the second fluorescent labeling compound and about 240 μsec for the target protein (FKBP) with which both the first fluorescent labeling compound and the second fluorescent labeling compound interacted.

Since each sample can be analyzed in a few seconds to a minute, it was possible to measure very quickly. As a result, the compound with Kd = 0.8 mM (Compound 3 shown in FIG. 6) was hit.

FIG. 7 shows the data (autocorrelation function) of the hit compound 3 obtained by the fluorescence correlation analyzer. This data is a complex in which two kinds of compounds interact (that is, a complex of the target protein, compound 2 and compound 3).
It is the analysis data of the autocorrelation function for calculating the translational diffusion time of. The horizontal axis represents time (milliseconds) and the vertical axis represents the autocorrelation function. The dotted line shows the data detected by the 543 nm wavelength laser, and the solid line shows the data detected by the 633 nm laser.

Thus, using the translational diffusion time of the complex in which two kinds of compounds interacted with each other was about 240 μsec, the concentration of each molecular species (concentration of the fluorescent labeled compound in the free state, the interaction with The concentration of the complex) was calculated. In this way, the Kd value (dissociation constant) as an index of affinity was determined.

Further, the compound 3 in which the side chain of the compound 3 is changed
9 to 9 were prepared, and the Kd value (dissociation constant) of each was determined. It was found that compound 9 binds to the target protein most stably.

Compound 2 (Kd = 2 μM) and Compound 9 (Kd
A more stabilized complex can be obtained by covalently binding (d = 100 μM) with an appropriate linker.
From the chemical structure, it can be seen that the methyl ester group of compound 2 and the benzoyl group of compound 9 are close in the complex state. Therefore, when 5 kinds of compounds were synthesized by using methylene groups having different lengths as linkers, all exhibited dissociation constants of the order of nM. It was found that among these 5 kinds of compounds, the compound having the highest affinity for FKBP was the compound 10 and Kd = 19 nM.

[0100]

As described above, according to the screening method of the present invention, it is possible to solve all the problems of the conventional techniques using NMR. In particular, the method of the present invention can measure the interaction at the molecular level more quickly and inexpensively when examining the interaction between a compound and a biomolecule (eg, receptor). Also, conventional NMR
According to the structure-activity relationship according to, the protein could be analyzed only up to a domain as small as 20 kDa, but the method of the present invention can also analyze a protein of 100 kDa or more.

[Brief description of drawings]

FIG. 1 is a diagram showing a cycle of drug design.

FIG. 2 is a diagram showing an outline of SAR by NMR.

FIG. 3 is a diagram illustrating an outline of fluorescence intensity distribution analysis (FIDA).

FIG. 4 is a diagram showing an outline of an example in which the screening method of the present invention is applied to drug discovery screening.

FIG. 5 is a diagram showing an example of a fluorescence correlation analyzer used in the screening method of the present invention.

FIG. 6 shows FK using the screening method of the present invention.
The figure which shows the outline of the example which searched the inhibitor of 506 binding protein (FKBP).

FIG. 7 is a view showing data (autocorrelation function) of single molecule fluorescence analysis of a substance capable of binding to a receptor.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Laser light source, 2 ... Light intensity adjusting means, 3 ... Light attenuation rate selecting device, 4 ... Dichroic mirror, 5 ... Objective lens,
6 ... Stage, 7 ... Filter, 8 ... Tube lens, 9
... Reflector, 10 ... Pinhole, 11 ... Lens, 12 ... Photodetector, 13 ... Fluorescence intensity recording means

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/566 G01N 33/566 F term (reference) 2G043 AA01 CA04 DA02 EA01 FA02 GA02 GB01 GB18 GB19 HA01 HA02 HA09 JA03 LA01 2G045 FA12 FA16 FA29 FB07 FB12 GC15 2G054 AB10 EA03 GA04 GA05

Claims (9)

[Claims] 1. A method for screening a substance capable of optimally binding to a receptor by single molecule fluorescence analysis, which comprises a step of searching for a substance capable of binding to the receptor and binding to the receptor obtained by said step. Performing the structural optimization of possible substances. 2. A method for screening a substance capable of binding to a receptor having a first binding site and a second binding site by single molecule fluorescence analysis, the substance being capable of binding to the first binding site. And a step of searching for a substance capable of binding to the second binding site under the condition that the substance capable of binding to the first binding site obtained in the above step binds to the receptor A method comprising the steps of: 3. A method for screening a substance capable of optimally binding to a receptor having a first binding site and a second binding site by single molecule fluorescence analysis, the method being capable of binding to the first binding site. A step of searching for a substance, a step of optimizing the structure of a substance capable of binding to the first binding site obtained by the step, and an optimal binding to the first binding site obtained by the step A substance capable of binding to the second binding site under the condition that the substance binds to the receptor, and a substance capable of binding to the second binding site obtained by the process And the step of optimizing the structure. 4. The method according to claim 3, wherein the substance is capable of optimally binding to the first binding site and the substance capable of optimally binding to the second binding site. The method further comprising the step of preparing a substance capable of optimally binding to the receptor by screening for the length and structure of the receptor. 5. A substance capable of binding to a receptor having n (n is an integer of 2 or more) binding sites from the first binding site to the nth binding site is screened by single molecule fluorescence analysis. A method of searching for a substance capable of binding to a first binding site, comprising: (1) from a first binding site to a (X-1) th binding site;
1) The step of searching for a substance capable of binding to the X-th binding site under the condition that each substance capable of binding to one binding site binds to the receptor, from X = 2 to X = n Repeating (n-1) times in sequence. 6. A substance capable of optimally binding to a receptor having n (n is an integer of 2 or more) binding sites from the first binding site to the nth binding site by single molecule fluorescence analysis. A method for screening, comprising a first step of searching for a substance capable of binding to a first binding site, and optimization of the structure of a substance capable of binding to the first binding site, obtained by the first step. And a second step from the first binding site to the (X-1) th binding site (X-
1) The third step of searching for a substance capable of binding to the Xth binding site under the condition that each substance capable of binding to one binding site is optimally bound to the receptor, and the third step A fourth step of optimizing the structure of the substance capable of binding to the X-th binding site obtained by the step, wherein the third step and the fourth step are X = 2 to X = n. Up to (n-1) times in sequence. 7. The method according to claim 6, wherein n substances are selected from a substance capable of optimally binding to the first binding site to a substance capable of optimally binding to the nth binding site, The method further comprising the step of preparing a substance capable of optimally binding to the receptor by screening for the length and structure of the linker for connecting each. 8. The substance capable of binding to the receptor is a target drug in drug discovery.
The method according to any one of 1 to 7. 9. The method according to claim 1, wherein the substance capable of binding to the receptor is labeled with a photodetectable substance.