JP2002241397A - Fluorescent protein and method for searching medicine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for searching a medicine, in which fluorescent light from an excited state of a fluorescence resonance energy transfer is used to observe an interaction between biological molecules and search the medicine. SOLUTION: This method for searching the medicine comprises observing the fluorescent light from the excited state of the fluorescence resonance energy transfer. In the method for searching the medicine, the following fluorescent protein FtatRhd is used. The fluorescent protein has Tat49-57 peptide. The fluorescent protein has fluorescein and tetramethylrhodamine as fluorochromes. When bound to HIV-1 TAR RNA, the fluorescent protein emits fluorescent light from the excited state of the fluorescence resonance energy transfer. The fluorescent protein quenches in the molecule in a free state.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a fluorescent protein (including a polypeptide) which enables a highly efficient drug search method.
And a drug search method using the fluorescent protein.

[0002]

2. Description of the Related Art At present, many high-throughput screening methods (HTS) are used in reporter gene assays.
This is a biochemical method, which is to observe the target reaction by replacing it with a spectroscopic signal.

[0003] Depending on the type of screening, there is an advantage that it can be observed at the same time as cytotoxicity. However, since the target reaction is viewed indirectly, various sub-signals are converted in the course of conversion to a spectroscopic signal. (error or noise)
Was included.

[0004] Therefore, it cannot be denied that the compounds obtained as a result of the screening may contain a large amount of compounds that do not meet their intended purpose (substances that nonspecifically bind to the target). In addition, there are many interactions between biomolecules that cannot be detected by a method using fluorescence quenching or fluorescence anisotropy by labeling with a single dye.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made in view of such problems, and biomolecules which have been considered to be difficult by the conventional method, the fluorescence intensity change method, and the fluorescence anisotropy change method, Alternatively, targeting the receptor and its ligand, fluorescence resonance energy transfer
nsfer, FRET), internal charge transf
er, ICT), which includes excimers, exciplexes,
Metal-centered charge
transfer, MCT), intramolecular torsional charge transfer (twistedintra
molecular charge transfer (TICT), and aims to develop a method for observing the interaction between biomolecules and searching for drugs using fluorescence from these specially excited states.

[0006]

Means for Solving the Problems The fluorescent protein of the present invention has a drug search function. The fluorescent proteins described above may have one or more fluorescent dyes. Further, the above-described fluorescent protein may have a flexible structure. The above-mentioned fluorescent protein may have a Tat49-57 peptide in some cases. When the above-mentioned fluorescent protein binds to RNA, it may emit fluorescence from the excited state of fluorescence resonance energy transfer. In addition, the above-mentioned RN
A may be messenger RNA. Also,
The above-mentioned fluorescent protein, when released, may cause intramolecular quenching. The above-mentioned fluorescent protein may have fluorescein and tetramethylrhodamine as fluorescent dyes in some cases.

[0007] The drug search method of the present invention is based on a fluorescence analysis method. In the drug discovery method described above, the fluorescence analysis method may include fluorescence from the excited state of fluorescence resonance energy transfer, internal charge transfer or excimer, exciplex, charge transfer within a metal chelate, or intramolecular torsional charge transfer. is there. In the above-described drug search method, the above-described fluorescent protein may be used.

According to the fluorescent protein and drug search method of the present invention, the following results are obtained. In other words, the fluorescent protein forms a dye-dye dimer due to its flexible structure, the peptide bends when released, and the hydrophobic interaction. As a result, intramolecular quenching occurs within the fluorescent protein. In addition, when this fluorescent protein binds to RNA, the bent structure is elongated to dissolve the dye-dye dimer, and fluorescence resonance energy transfer occurs.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention relating to a fluorescent protein and a drug search method will be described. The invention according to the present embodiment is to create a fluorescent protein having drug discovery ability, and to construct a system for directly observing and finding a compound that binds to a target biomolecule using fluorescence resonance energy transfer or charge transfer fluorescence. Aim.

The invention according to the present embodiment is summarized as follows. First, a biomolecule, protein, nucleic acid (DNA, RNA) on which a drug to be searched acts and a biomolecule (ligand) on which these act on are labeled with a fluorescent dye and tracer. Create a molecule. Then, the change in the molecular structure caused by the binding between the biomolecules is converted into a spectral signal using fluorescence resonance energy transfer or charge transfer fluorescence, and the biomolecules, mainly proteins and genetic information that controls biological information. Nucleic acid
We have developed a method (High-throughput screening, HTS) for rapidly and abundantly searching for molecules that bind to (DNA, RNA).

That is, a biomolecule serving as a ligand is labeled with one or more fluorescent dyes, and emission or charge-transfer fluorescence from fluorescence resonance energy transfer generated upon binding between the molecules is observed. The presence of a binding inhibitor between these two molecules is observed as a significant increase or decrease in luminescence or charge transfer fluorescence from fluorescence resonance energy transfer due to competitive binding to conventional ligand biomolecules.

The invention of the drug search method according to the present embodiment is a drug search method based on fluorescence analysis. Here, the specific contents of the fluorescence analysis method are as follows. That is,
Focusing on the fact that many biomolecules, such as proteins and nucleic acids, greatly change their structures as they bind, the biomolecules that serve as ligands are labeled with two fluorescent dyes or multiple luminescent factors, and when binding between molecules, The resulting fluorescence resonance energy transfer (F)
RET), internal charge transfer (ICT)
That is, charge transfer within excimers, exciplexes, and metal chelates (Metal-centered charge transfer, M
CT), twistedintramolecular c
This is a method of observing fluorescence from a special excited state such as harge transfer, TICT).

This search method can be designed for a wide range of biomolecules and can be immediately applied to drug search.

Next, the invention according to the present embodiment will be specifically described. As a specific application example of the present invention,
Messenger RNA (TAR RNA, FIG. 1), which is a transcriptional active site of the genomic information of HIV-1 virus, and a protein (Tat) bound thereto were taken up, and the effectiveness of the present invention was proved. In the case of a Tat protein that binds to TAR RNA, it does not have to wait for a rigid structure, that is, it has a flexible structure, so that the fluorescence depolarization method cannot be used.

Therefore, the RNA binding site of the Tat protein, Ta
C-terminal of t49-57 peptide is tetramethylrhodamine,
Fluorescein-labeled fluorescent peptide at the N-terminus (Fta
tRhd, Fig. 1) was chemically synthesized, and fluorescence resonance energy transfer (FRET) from fluorescein to tetramethylrhodamine was performed.
The tracer (FtatRhd) of the high-throughput screening method (HTS) utilizing was prepared.

Tat49-57 which is an mRNA binding site of Tat protein
A polypeptide consisting of 15 amino acids in which three alanines are arranged at both ends of the peptide as spacers was synthesized by a solid phase synthesis method using Fmoc-protected amino acids, and 5-carboxyfluorescein succinimide (Molecular Prob
fluorescein unit is introduced by a condensation reaction to form an amide bond at the N-terminus, and the rhodamine unit is attached to the C-terminal cysteine via a thioether bond using tetramethylrhodamine maleimide (manufactured by Molecular Probes). Introduced.

This was purified by high-resolution liquid chromatography (HPLC) using a reversed-phase column, and mass spectrometry (MALDI
-TOF MS).

A 100 nanomolar aqueous solution of FtatRhd is prepared in a buffer solution (20 millimolar Tris-HCl, 140 millimolar sodium chloride, 5 millimolar potassium chloride, 1 millimolar magnesium chloride, pH 7.5), and titration with a TAR RNA solution results in significant FRET. An increase in fluorescence was observed (FIGS. 3 (B) and (C)).

That is, natural HIV-1 TAR RNA (wtTAR)
The binding of the Tat protein is converted to a FRET fluorescence signal by using the tracer FtatRhd. This mechanism is shown in FIG.
This will be described with reference to FIG. Tracer (FtatRhd)
In a simple substance (in a free state), the dye is bent due to the flexibility of its structure, and the dyes arranged at both ends come close to each other due to hydrophobic interaction, and a dimer of the dye is formed in the molecule. As a result, the excited state of the donor (D) is intramolecularly quenched by the acceptor (A). However, when FtatRhd binds to wtTAR, a distance is maintained between the two dyes, and light emission accompanied by fluorescence resonance energy transfer is observed.

The TTA protein binding site of wtTAR, in which three bases essential for binding recognition have been removed (this is already known not to bind Tat protein; dTAR, FIG. 2)
Even when added to a 100 nanomolar aqueous solution of FtatRhd, no change was observed in the fluorescence spectrum of FtatRhd (FIG. 3 (C)). This result indicates that FtatRhd is HIV-1TAR RNA
Has been shown to specifically bind to

On the other hand, Tat labeled only with fluorescein
When the buffer solution (Ftat, FIG. 2) was similarly titrated with TAR RNA, only a 18% decrease in fluorescence was observed (FIG. 4).
(A)). Labeled with tetramethylrhodamine (tat
Rhd (FIG. 2) showed no change in fluorescence even when TAR RNA was dropped (FIG. 4 (B)).

In order to prove that this fluorescent protein having drug discovery ability functions, TAR RNA of Tat protein was used.
The natural-type Tat49-57 peptide (wt-Tat) which is a binding site and a model peptide (T81, T71, T61 T51, T5) in which arginine necessary for specific binding is substituted with alanine from this peptide
2, T41, T31, Table 1) were prepared, and the drug search ability of FtatRhd was evaluated.

FtatRhd 100 nanomolar, TAR RNA 80
An aqueous solution of 0 nanomolar is prepared in a buffer solution (pH 7.5), and the solution is titrated with a solution of the drug.
It is observed as a decrease in FRET fluorescence reflecting the binding force to (FIG. 5).

The binding force (dissociation constant) of the native type and the model peptide to TRA RNA can be calculated by equation 1. (Formula 1) [Drug] 0 = (KD (i1-i) / Kd (i-i0) + 1
) × ([target] 0-Kd (i-i0) / (i1-i)-[tracer] 0 (i-i0) / (i1-i0))

Here, [drug] 0, [target] 0, and [tracer] 0 are the added drug, target (HIV-1 TAR RNA in this embodiment, FIG. 1), and tracer (this embodiment, respectively). Then, the concentration of FtatRhd, Fig. 1), KD and Kd are the dissociation constants of drug and target, tracer and target, i1, i0, i,
Are the target and tracer complex, tracer,
It represents the fluorescence intensity at an observation wavelength of 576 nm observed when a drug is added to a mixed solution of a target and a tracer.

Table 1 shows the results. The dissociation constant of the wt-Tat peptide was determined to be 70 nanomolar, indicating that the dissociation constant increases as the number of arginines substituted with alanine increases, indicating that the binding force to wtTAR RNA is weak. The above results indicate that FtatRhd certainly functions as a drug discovery tracer targeting HIV-1 TAR RNA, and by utilizing this principle, the ability of various drugs to inhibit the binding of HIV-1 TAR RNA to the Tat protein complex can be easily determined by fluorescence observation. It is shown that it can be converted into a numerical value.

[0027]

[Table 1]

The results of evaluating this search method using existing antibiotics are shown below. The antibiotics to be detected were gentamicin, hygromycin B, kanamycin A, kanamycin B, neomycin, neamine, paromomycin, ribostamycin, streptomycin, and tobramycin, and the concentration at the time of observation was 50 micromolar. And the rates of change in fluorescence intensity at that time were compared (FIG. 5).

Here, I, I0, and IT are FtatRhd 100 micromolar solution and TAR RNA 800 in the presence of TAR RNA 800 micromolar when 50 micromolar of drug is added, respectively.
Observation wavelength of FtatRhd 100 micromolar solution in the presence of micromolar, FtatRhd 100 micromolar solution only
Indicates the fluorescence intensity at 576 nm.

If (I0-I) / (I0-IT)> 0.5, tobramycin, paromomycin, neamine, neomycin, kanamycin B, kanamycin A and gentamicin are detected, but (I0-I) / (I0-IT) -IT)> 0.7
5 means tobramycin, neamine, neomycin,
Kanamycin B is detected. Also, (I0-I) / (I0 -I
At T)> 0.90, only neomycin is detected.

The natural Tat protein is (I0-I) / (I0
A concentration of about 1/1000 of neomycin (50 nanomolar) is sufficient to show -IT)> 0.75, and the concentration of the specimen to be prepared and the change rate of the fluorescence intensity indicated at that time (I0-I) / (I0
-IT) can be used to search for a suitable drug. The fluorescent screening method targeting HIV-1 TAR RNA is a large-scale and rapid screening method using a 96-well plate (high-throughput screen).
ening, HTS), and is expected to contribute to the development of anti-HIV drugs.

In the above-described embodiment, an example has been described in which the Tat49-57 peptide and fluorescein and tetramethylrhodamine (fluorescent dye) are used as tracers. However, tracers are not limited to this. For example, the sequence can also use the Rev34-50 peptide for messenger RNA (RRE RNA) and its binding protein (Rev) that promote the transcriptional activity of genomic information of the same AIDS virus, HIV-1, and will generally be a tracer. The sequence of the peptide can be appropriately selected from the binding sites of the target biomolecule, protein, receptor and the like.

In addition, examples of the fluorescent dye include dansyl, dabsyl, coumarin, and the like, and a proper combination of these dyes can induce fluorescence resonance energy transfer.

The number of fluorescent dyes is not limited to two, but may be one or three to fifty.

In the above-described embodiment, a method utilizing fluorescence resonance energy transfer as a fluorescence analysis method has been specifically described. However, the fluorescence analysis method is not limited to a method using fluorescence resonance energy transfer.
Other methods of fluorescence analysis include internal charge transfer (internal
charge transfer (ICT), that is, charge transfer within excimers, exciplexes, and metal chelates.
ered charge transfer (MCT), intramolecular twisted charge transfer
(twisted intramolecular charge transfer, TICT), and the like.

The internal charge transfer is an excited state involving charge transfer in a dye molecule, and is a charge transfer from an excimer, an exciplex, a metal to a ligand (MLCT), or a charge transfer from a ligand to a metal (MLCT). LMCT), ligand-to-ligand charge transfer (LLCT), intramolecular torsional charge transfer (T
ICT).

In particular, excimer fluorescence is an excited state in which two identical dyes form a charge transfer dimer which is a more stable excited state.
dimer).

Also, exciplex fluorescence is an excitation complex (exclusion complex) formed by forming a charge transfer complex which is a more stable excited state formed from a dye and an amine.
It is light emission from the itedstate complex).

The charge transfer in a metal chelate is an intramolecular charge transfer that occurs when a metal ion and a suitable ligand form a complex (chelate), which includes the transfer of a metal to a ligand. Charge transfer (MLCT), ligand to metal charge transfer (LMCT).

In addition, intramolecular torsional charge transfer (TICT)
Is an excited state involving torsion and charge transfer of a dye molecule.

In the above-described embodiment, the field of exploring a large amount and a rapid search for molecules that bind to biomolecules, mainly proteins and nucleic acids (DNA, RNA), which are genetic information that controls biological information, has been described. However, the invention is not limited to this application. The present invention can be applied to other interactions between various biomolecules, including DNA and proteins or ligands, proteins and proteins, enzymes and substrates, receptors and ligands, etc. By selecting and designing fluorescent tracers, new drug discovery methods can be developed.

The present invention is not limited to the above-described embodiment, but can adopt various other configurations without departing from the gist of the present invention.

References Known: A high-throughput screening utilizing in
tramolecularfluorescence resonance energy transfer
for the discovery of themolecules that bind HIV-1
TAR RNA specifically Bioorganic & Medicinal Chemis
try Letters 2000, 10, 1857-1861 (2000.8.21presse
d)

Presentation at the conference: Development of a novel search method for molecules that bind to HIV-1 TAR RNA using intramolecular fluorescence resonance energy transfer Chiharu Matsumoto, Keita Hamasaki, Hisakazu Mihara, Akihiko Ueno The 15th Symposium on Biological Science, 2000 , 9.23-24, Nara Women's University

[0045]

The present invention has the following effects. Unlike the conventional method using the reporter gene, fluorescence resonance energy transfer (fluorescence resona
nce energy transfer, FRET), internal charge transfer (interna
lcharge transfer, ICT) charge transfer within excimers, exciplexes, and metal chelates (metal-ce
ntered charge transfer (MCT), intramolecular torsional charge transfer
(twisted intramolecular charge transfer, TICT), which utilizes fluorescence from a special excited state, converts information on the binding force of a drug that binds to a specific target into spectroscopic information. Information can be observed directly. Also, since it can be easily digitized, high-t
It can be applied to hroughput screening (HTS), making it easier to compare drug effects.

[Brief description of the drawings]

FIG. 1 is a diagram showing the double structure of native HIV-1 TAR RNA (wtTAR) and the amino acid sequences of fluorescent Tat and FtatRhd and the structure of a dye.

FIG. 2: Double structure of dTAR RNA that does not bind to Tat protein,
FIG. 3 is a diagram showing the amino sequence of fluorescent Tat, Ftat, and tatRhd and the structure of a dye.

Fig. 3 Tracer (FtatRhd) and natural TAR RNA (wtTAR)
And the resulting structural change of FtatRhd (A), the change in fluorescence spectrum when natural TAR RNA (wtTAR) is added to an aqueous solution of tracer (FtatRhd) (B), the tracer
FIG. 4 is a diagram showing a comparison of fluorescence intensity at 576 nm when natural TAR RNA (wtTAR) or non-binding TAR RNA (dTAR) is added to an aqueous solution of (FtatRhd).

FIG. 4 is a diagram showing a fluorescence spectrum when TAR RNA is added to an aqueous solution (100 nanomolar) of Ftat (A) and tatRhd (B).

Fig. 5 Tracer (FtatRhd) 100 nanomolar and natural TA
R RNA (wtTAR) Changes in fluorescence spectrum when drug is added to 800 nanomolar aqueous solution (A), natural TAR RNA (w
tTAR) and drug binding, accompanying structural change of FtatRhd (B), tracer (FtatRhd) 100 nanomolar and native
FIG. 4 is a diagram showing a comparison of fluorescence intensity at 576 nm when a drug is added to an aqueous solution of 800 nm TAR RNA (wtTAR).

FIG. 6 is a diagram showing search results using antibiotics as an example.

Claims (12)

[Claims] 1. A fluorescent protein having a drug search function. 2. The fluorescent protein according to claim 1, which has one or more fluorescent dyes. 3. The fluorescent protein according to claim 1, which has a flexible structure. 4. The fluorescent protein according to claim 1, 2 or 3, has a Tat49-57 peptide. 5. The fluorescent protein according to claim 1, 2, 3, or 4, emits fluorescence from an excited state of fluorescence resonance energy transfer when bound to RNA. 6. The RNA according to claim 5, which is a messenger RNA. 7. The fluorescent protein according to claim 5 or 6 causes intramolecular quenching when released. 8. The fluorescent protein according to claim 2, 3, 4, 5, 6, or 7 has fluorescein and tetramethylrhodamine as fluorescent dyes. 9. A drug search method based on a fluorescence analysis method. 10. The drug search method according to claim 9, wherein
Fluorescence analysis means fluorescence resonance energy transfer, excimer,
Includes fluorescence from excited states of exciplexes, internal charge transfer, charge transfer from metal to ligand, or intramolecular torsional charge transfer. 11. The drug search method according to claim 9, wherein
Fluorescence analysis includes fluorescence from the excited state of fluorescence resonance energy transfer. 12. The drug search method according to claim 9, 10, or 11, wherein the drug search method according to claim 1, 2, 3, 4, 5, 6,
The fluorescent protein described in 7 or 8 is used.