JP2001322999A - Derivative of physiologically active substance having receptor recognition site preserved and method of using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of deactivating the physiological activity of the physiologically active substance, as the receptor-recognizing site of the physiologically active substance is retained, the deactivation derivative therefor, the use thereof, the derivative of the physiologically active substance of which the physiological activity is retained even after the chemical modification and the use thereof. SOLUTION: In the substance that manifests its physiological activity, when it binds with the action site in the receptor, linkers are allowed to link with the other parts than the sites recognizing the receptor, a substance that cannot invade into the receptor or a substance that has high affinity to the substance that cannot invade into the receptor are linked to the other chain end whereby the physiologically active substance is deactivated as the sites of recognizing the receptor for the physiologically active substance are retained. Further, this invention relates to the deactivation derivative, a substance that can immobilize the other end of the linker to the carrier, a substance that can immobilize the other end of the linker to the carrier or the derivative of the physiologically active substance of which the physiological activity is retained prepared by linking the substance immobilized to the carrier to the substance having the affinity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for inactivating the physiological activity of a physiologically active substance while preserving the receptor recognition site of the physiologically active substance, a non-activated derivative thereof, an antibody thereto, and use thereof. About the method. The present invention also relates to a derivative of the physiologically active substance while maintaining the physiological activity, and a method for using the same. More specifically, the present invention provides
In a substance that expresses a biological activity by binding to an action site inside a receptor, a linker is bound to a portion other than the receptor recognition site of the physiologically active substance, and the other end of the linker enters the receptor. A method of inactivating a biologically active substance while preserving a receptor recognition site of the biologically active substance, comprising binding a substance having an affinity to a substance which cannot be invaded or a substance which cannot enter the receptor. Non-activated derivative, an antibody thereto, and its use, and chemically modifying a portion other than the receptor recognition site of the physiologically active substance, binding a linker to the chemically modified site,
The other end of the linker is immobilized on a carrier, or a substance capable of being immobilized on (or having affinity with) the carrier or a substance having affinity with the substance immobilized on the carrier is immobilized on the other end of the linker. The present invention relates to a derivative of a physiologically active substance having a physiological activity, and a method of using the same.

[0002]

2. Description of the Related Art Animals such as snakes, scorpions, and shellfish sometimes have toxins, and these toxins may inhibit the action of nerve cells to immobilize or kill other animals. Many of these toxins are known to act on cellular receptors (including channels and the like). Vaccines against these toxins have been produced, but it is often difficult to produce antibodies against these toxins themselves. This is because when sensitizing animals to these toxins to produce antibodies, the sensitized animals often die before making antibodies.
For example, μ-conotoxin isolated from potato shellfish is a peptide toxin that selectively inhibits sodium ion channels in cells such as muscle, but it has also been difficult to produce antibodies for these.

[0003] Conventionally, in order to suppress the physiological activity of a physiologically active substance, it has been practiced to focus on the physiologically active site and modify the site by chemically modifying the site. . For example, in the case of the above-mentioned toxins, denaturation has been performed by denaturing physiologically active sites, for example, by denaturing by heating. But,
When the bioactive site is chemically or physically changed, since the bioactivity itself of the bioactive substance is changed,
Antibodies to it cannot be produced.

Further, when it is desired to measure the depth of the site of action of a receptor on which a physiologically active substance acts, the size of the entrance of the receptor, etc., the three-dimensional structure of the receptor must be determined. And a complicated operation was required.

SUMMARY OF THE INVENTION In order to solve the conventional problems, the present invention deactivates the physiological activity of a physiologically active substance while preserving the receptor recognition site of the physiologically active substance. The present invention provides a method, a non-activated derivative thereof, and a derivative of a physiologically active substance which retains its biological activity even after chemical modification. The present invention also provides a novel method for measuring the size of the receptor entrance and the depth of the site of action, and further provides a method for isolating a new sodium channel or calcium channel. But also.

[0005]

Means for Solving the Problems The present invention relates to a substance which expresses a physiological activity by binding to an internal action site of a receptor, wherein a linker is bound to a portion other than the receptor recognition site of the physiologically active substance, The other end of the linker preserved a receptor recognition site of a physiologically active substance comprising binding a substance that cannot enter the receptor or a substance that has an affinity with a substance that cannot enter the receptor. The present invention relates to a method for inactivating the physiologically active substance as it is, an inactivated derivative thereof, an antibody thereto, and a method for producing the same. Further, the present invention provides a method of treating a cell with a non-activated derivative of the above-mentioned physiologically active substance, in which a cell having an affinity for a substance that cannot enter a receptor at one end of a linker is bound. Measuring the activity of the receptor by bringing the receptor recognition site of the active substance into a state capable of binding to the receptor, and then treating the cell line with a substance that cannot enter the receptor, and measuring the activity of the receptor And a method for measuring the affinity of a biologically active substance, treating cells with a non-activated derivative of the aforementioned biologically active substance to which a substance that cannot enter the receptor at one end of the linker is bound,
A method for measuring the distance between the entrance of the receptor and the site of action of the physiologically active substance, comprising measuring the activity of the receptor, and a method of linking a cell with a linker having a length that does not reach the site of action of the receptor. One end is treated with a non-activated derivative of the physiologically active substance having a substance corresponding to the size of the receptor entrance bound thereto, and the activity of the receptor is measured. It relates to a method for measuring or measuring the properties of a receptor, such as a method for measuring size. Furthermore, the present invention provides a substance that expresses a biological activity by binding to an internal action site of a receptor, wherein a portion other than the receptor recognition site of the physiologically active substance is chemically modified, and a linker is added to the chemically modified site. Bind, and fix the other end of the linker to the carrier,
Alternatively, a physiologically active substance having retained biological activity, comprising a substance that can be immobilized on a carrier (or has an affinity with a carrier) or a substance that has an affinity with a substance immobilized on a carrier is bonded to the other end of the linker. And derivatives thereof. Furthermore, the present invention relates to a method for isolating a sodium channel or a calcium channel using a derivative of a physiologically active substance having the physiological activity.

The outline of the present invention will be described using an ion channel as an example of a receptor. FIG. 1 shows a state in which a physiologically active substance 4, in this example, a physiologically active substance 4 acting as a toxic substance, is bound to an ion channel 1 provided through a cell membrane 2 to an action site 3 of the ion channel 1. This is schematically shown. The receptor recognition site 5 of the physiologically active substance 3
By binding to the action site 3 of the ion channel 1 for the physiologically active substance, the ion channel 1 is blocked, and ions enter the cell and disappear, resulting in abnormalities in the cell.

FIG. 2 shows one embodiment of the present invention. The biologically active substance 4 is bonded to the substance 7 which cannot enter the receptor by the linker 6, and the receptor recognition site 5 of the biologically active substance 4 and the ion channel 1 Cannot bind to the action site 3, and as a result, the ion channel 1 cannot be blocked by the physiologically active substance 4, so that ions can freely pass through the ion channel 1. As a result, the activity of the physiologically active substance 4 on the ion channel 1 is deactivated although the receptor recognition site 5 retains its active chemical structure.

FIG. 3 shows another embodiment of the present invention. The physiologically active substance 4 is bound by a linker 6 to a substance 8 having an affinity for a substance which cannot enter the receptor, and this substance 8 binds to the substance 7 which cannot enter the receptor by affinity. ing. As in the case of FIG. 2 described above, the linker recognition site 5 of the physiologically active substance 4
And the active site 3 of the ion channel 1 cannot be bound, and as a result, the ion channel 1 cannot be blocked by the physiologically active substance 4, and ions can freely pass through the ion channel 1. As a result, bioactive substance 4
The activity of the ion channel 1 is deactivated despite that the receptor recognition site 5 retains its active chemical structure. In FIGS. 2 and 3, when the linker is sufficiently long (for example, Compound 6 described later)
) Naturally shows no activity inhibition and shows the original activity.

Next, the present invention will be described in more detail by using μ-conotoxin isolated from potato shellfish as a physiologically active substance. It is known that μ-conotoxin, which is a peptide toxin, is a toxin acting on site 1 of a sodium channel and is the same type of poison as tetrodotoxin and saxitoxin. FIG. 4 shows the outline of the amino acid sequence of μ-conotoxin and its three-dimensional structure. FIG. 4 shows μ-conotoxin GIIIA, which consists of 22 amino acids from arginine at position 1 to alanine at position 22. Cysteine at position 3 and cysteine at position 15, 4
Cysteine at position 20 and cysteine at position 20, and cysteine at position 10 and cysteine at position 21 are each bonded by an SS bond. The active center of μ-conotoxin GIIIA is near arginine at position 13.

Each amino acid of μ-conotoxin GIIIA
The effect on bioactivity can be determined by converting each amino acid to alanine or lysine.
FIG. 5 shows the result of the examination by substituting the above. In FIG.
The contraction of the longitudinally isolated rat diaphragm specimen by electrical stimulation was reduced by 50%.
% Inhibitory concentration (IC₅₀(Log (M))),
The dotted line indicates the IC of natural μ-conotoxin GIIIA.₅₀Shows
are doing. The horizontal axis in FIG. 5 is the amino acids from position 1 to position 22
In FIG. 5, black amino acids represent amino acids at the corresponding site by alanine.
IC when replaced with₅₀And gray are the
IC when amino acid is replaced with lysine₅₀Shows
You. According to FIG. 5, for example, arginine at position 13 is replaced with alanine.
IC for lysine and lysine ₅₀Rise, that is, no activity
Arginine at position 13 is μ-conotoxin G
It is thought that it forms an essential part of the physiological activity of IIIA
It is. On the other hand, threonine at position 5 and lysine at position 9
Can be replaced with alanine_{5 0}There is no change in this
This site has an effect on the physiological activity of μ-conotoxin GIIIA.
It is considered to be a part that does not affect the sound.

The present inventors have focused on the active site at position 13, ie, threonine at position 5, which corresponds to the sterically almost opposite side of the receptor recognition site. The threonine site at position 5 is a portion that sterically corresponds to the back of the receptor recognition site and does not affect the biological activity. The present inventors substituted the amino acid with a functional group in order to introduce a linker at the threonine position at position 5. In this experiment, cysteine or lysine was employed as the amino acid having a functional group. μ-conotoxin GIIIA has lysine at the 8th and 9th positions, etc. In order to clarify the chemical reactivity with the amino group of lysine, cysteine having a mercapto group is used as an amino acid having a functional group. preferable.

The natural μ-conotoxin GIIIA (μGIII
A) μ-conotoxin GIIIA in which threonine at position 5 has been replaced with S-methylbenzylcysteine (Cys (Me
Bzl) 5), and μ-conotoxin GIIIA substituted at position 5 with N-biotinylated lysine (Lys (Biot)
Regarding 5), the contraction of rat skeletal muscle was tested. FIG. 6 shows the results. FIG. 6 is a photograph instead of a drawing showing the results of a skeletal muscle contraction experiment. The vertical axis in FIG. 6 shows the skeletal muscle tension (mN (millinewton)). The large black portion indicates that the sodium ion channel is in a normal state and the skeletal muscle has a sufficient tension. Is shown. The horizontal axis in FIG. 6 is time (minute). The upper part of FIG. 6 shows the transition of the tension when 0.3 μM of natural μ-conotoxin GIIIA was added. μ-conotoxin GIII
The addition of A rapidly decreased the tension, indicating that abnormalities occurred in the skeletal muscle. After the tension has been removed, this is washed (white arrow in FIG. 6).
It can be seen that the added μ-conotoxin GIIIA was washed away and the tension was recovered. At this time, the inhibition rate of 0.3 μM was 99%.

FIG. 6 shows a μ-conotoxin GIIIA (Cy) in which the 5-position is substituted with S-methylbenzylcysteine.
s (MeBzl) 5) was added at 0.3 μM, and 1 μm was added.
The time when M was added is shown. Since the tension is lost, it can be seen that this has the same physiological activity as the natural one. Recovery of tension is seen by washing with water (open arrows in FIG. 6) as in the case of natural products. At this time, the inhibition rate of 0.3 μM was 47%. The lower part of FIG.
The figure shows the case where 0.3 μM of μ-conotoxin GIIIA (Lys (Biot) 5) in which the 5-position was substituted with N-biotinylated lysine and then 1 μM were further added. Since the tension is lost, it can be seen that this has the same physiological activity as the natural one. Recovery of tension is seen by washing with water (open arrows in FIG. 6) as in the case of natural products. At this time, the inhibition rate of 0.3 μM was 62%.

Thus, even if a portion other than the receptor recognition site of a physiologically active substance is subjected to simple chemical modification, the physiological activity of the physiologically active substance cannot be inactivated. This is because the portion other than the receptor recognition site of the physiologically active substance does not greatly contribute to its physiological activity. Next, the present inventors used avidin having an affinity for biotin, and substituted μ-conotoxin GIIIA (Lys (Bi
ot) An experiment for 5) was performed. FIG. 7 shows the results. FIG. 7 is a photograph instead of a drawing showing the results of a skeletal muscle contraction experiment. The vertical and horizontal axes in FIG. 7 are the same as those in FIG. FIG. 7A (the uppermost row in FIG. 7) shows Lys (Biot) 5.
And Avidin (Av) are added simultaneously. Initially, 1 μM was added, but no abnormality in skeletal muscle contraction was observed. Next, even if the concentration was increased to 3 μM or 10 μM, no physiological activity by μ-conotoxin GIIIA could be observed. In the experiment shown in the lower part of FIG. 6, Lys (Biot) 5 showed a 62% inhibition rate at 0.3 μM, and showed about the same level of inhibition as the natural μ-conotoxin GIIIA at 1 μM. Was found to be able to be inactivated even at a concentration of 10 μM.

Next, 1 μM of avidin (Av) was added first, and then 1 μM of Lys (Biot) 5 and 3 μM of Lys (Biot) 5.
7b (from the top in FIG. 7 to FIG. 7).
(Stage). As a result, it was found that the activity of Lys (Biot) 5 was not expressed even if avidin was previously added. The right end of FIG. 7b, the right end of FIG. 7c, and the d of FIG. 7 show the results when natural μ-conotoxin GIIIA was added for comparison. From the results on the right end of FIG. 7B, in the case of natural μ-conotoxin GIIIA, even if avidin is present, there is almost no effect on its biological activity, and the biological activity is almost the same as when avidin is not present. It turns out that it expresses. Further, c in FIG. 7 (third stage from the top in FIG. 7) shows the result when Lys (Biot) 5 was added at 1 μM first. Inhibition of skeletal muscle contraction due to the addition of Lys (Biot) 5 is observed. Then, 0.5 μM of avidin and 1 μM of avidin were added to the system.
It was found that inhibition of skeletal muscle contraction by ys (Biot) 5 gradually disappeared, and skeletal muscle contraction became normal.
When natural μ-conotoxin GIIIA was added to the normalized place, inhibition of skeletal muscle contraction was observed (right end of c in FIG. 7).

From the above experimental results, μ-conotoxin GIIIA (Lys) in which the 5-position was substituted with N-biotinylated lysine.
(Biot) 5) has the same inhibitory action on skeletal muscle contraction as the natural one, but avidin having affinity for biotin simultaneously with, before or after addition of Lys (Biot) 5 Was found to deactivate its inhibitory activity. And μ-conotoxin GII
IA is known to act on site 1 of the cell's sodium channel, which is in the middle of the channel, so that N-biotinylated lysine at position 5, which has the same physiological activity as the natural one, Μ-conotoxin G substituted with
Although IIIA (Lys (Biot) 5) is chemically modified, it binds to the site of action of the sodium channel and inhibits skeletal muscle contraction in the same manner as the natural one, but when avidin is added to it, Lys This means that the biotin moiety of (Biot) 5 bound preferentially to avidin and could no longer bind to sodium channels. Avidin is a giant protein having a molecular weight of about 65,000, and is a molecular species that cannot enter a small channel such as a sodium channel. Therefore, the physiologically active substance μ-conotoxin GIIIA can be obtained from just outside the channel.
Is caught by chemically modified biotin.

Furthermore, the following formula (I) is added to the mercapto group of μ-conotoxin GIIIA (Cys-5) in which threonine at position 5 is substituted with cysteine.

[0018]

Embedded image

The non-activated derivative A (biotin PEAC5 maleimide) to which biotinylated PEAC5 is bound and the μ-conotoxin GIIIA (Cys-5) in which threonine at position 5 is substituted with cysteine are attached to the mercapto group. , The following equation (II),

[0020]

Embedded image

A similar experiment was carried out using a non-activated derivative B (biotin PE maleimide) to which biotinylated PE was bound. FIG. 8 shows the results. FIG. 8 is a photograph instead of a drawing showing the results of a skeletal muscle contraction experiment. The vertical and horizontal axes in FIG. 8 are the same as those in FIG. Non-activated derivative A (biotin PEAC5 maleimide) is an example using a slightly longer linker, and non-activated derivative B (biotin PE maleimide) is an example using a shorter linker.
FIG. 8a (the top row of FIG. 8) first shows the non-activated derivative A
(Biotin PEAC5 maleimide) was added at 1 μM, and when contraction of skeletal muscle was inhibited, avidin (Av) was added at 1 μM.
The case where M was added is shown. The addition of avidin has gradually alleviated the inhibition of shrinkage, but the extent is relatively weak. FIG. 8b (the second row from the top in FIG. 8) shows that the non-activated derivative A (biotin PEAC5 maleimide) was 1 μM
And avidin are added at the same time. It can be seen that despite the presence of avidin, the activity of inhibiting skeletal muscle contraction is gradually increasing. FIG. 8c (the third row from the top in FIG. 8) shows first the non-activated derivative B (biotin P
E maleimide) was added at 1 μM, then 3 μM, and avidin (Av) was added at 1 μM when skeletal muscle contraction was inhibited. It can be seen that the addition of avidin has eased relatively quickly.

In the biotin-linker used for the non-activated derivative A (biotin PEAC5 maleimide),
Since the length of the linker portion is relatively long and biotin reaches near the entrance of the channel, the distance that can be caught even if avidin is added is small, and the dissociation between the physiologically active substance and the channel cannot be performed sufficiently, It is considered that rapid recovery of skeletal muscle contraction could not be observed even with the addition of avidin. On the other hand, in the case of the non-activated derivative B (biotin PE maleimide), the length of the linker portion is relatively short, and the linker portion is greatly caught by the addition of avidin. As a result, as shown in FIG. It is considered that the recovery of the contraction was observed.

It is considered that the results of these experiments are as shown in FIG. That is, biotin (8) is bound via a linker (6) of μ-conotoxin GIIIA ((4) in FIG. 3) which is a physiologically active substance. In the absence of avidin (7), the receptor recognition site (5) of μ-conotoxin GIIIA (4) and the site of action (3) of sodium channel (1) are bound, and sodium ions are introduced into cells. Of the skeletal muscle is consequently inhibited. Since the chemical modification of the present invention is characterized in that it is performed in a portion other than the receptor recognition site, it is natural that the chemical modification of the present invention does not greatly affect its physiological activity. is there. This is shown in the experiment shown in the lower part of FIG.
It is clear from the results of the first half of the experiment. When avidin (7) is present in this system, avidin (7) cannot enter the inside of channel (1) because of its large molecule, and as shown in FIG. Will be outside of the Then, Lys having a biotin moiety is determined by the affinity of biotin (8) and avidin (7).
(Biot) 5 looks as if it were entirely caught by avidin (7). As a result, the bond between the channel (1) and the physiologically active substance (4) is released, and the channel (1) can operate normally.

Furthermore, this indicates that the length of biotin and the linker is shorter than the distance from the entrance of the channel to the site of action. Because if the linker and biotin are longer than the distance from the channel entrance to the site of action, biotin can exit the channel and move bioactive substances in binding to avidin. Because it can be done freely outside the channel. As a result, the binding of avidin and biotin does not affect the biological activity. This indicates that the minimum length of the linker that does not change the biological activity is the distance between the receptor entrance and the site of action by measuring the biological activity by changing the length of the linker. ing. Therefore, according to this method, the distance between the site where the physiologically active substance is acting on the receptor and the entrance of the receptor can be measured by the length of the linker of the non-activated derivative of the present invention.

The fact that the whole Lys (Biot) 5 is caught by avidin indicates that avidin is of a size that cannot enter the receptor. Because if avidin was large enough to penetrate into the receptor, avidin would penetrate into the receptor to bind biotin, resulting in no change in the binding between the biologically active substance and the receptor. This is because the physiological activity is not affected. This means that by measuring the bioactivity of a bioactive substance using various substances of known sizes as substances that cannot enter the receptor in the non-activated derivative of the present invention, The size of the material at which the change occurs will be approximately comparable to the size of the receptor entrance. Therefore,
By this method, the size of the entrance of the target receptor can be measured.

Furthermore, since the bioactive substance bound to the site of action of the receptor is dissociated by the affinity of biotin and avidin, the biotin and the bioactive substance can be dissociated more than the binding force between the receptor and the bioactive substance. It can be seen that the affinity of avidin is greater. This means that the combination of "a substance having an affinity for a substance which cannot enter a receptor" and "a substance which cannot enter a receptor" of the present invention is changed to various substances whose affinity is known. This shows that the binding force between the receptor and the physiologically active substance can be measured. Therefore, this method of the present invention makes it possible to measure the binding strength between a receptor and a physiologically active substance.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a chemical modification of a physiologically active substance which enters a receptor and expresses its physiological activity. It is characterized in that it is performed from an inactive part. In particular, it is characterized by chemically modifying a physiologically inactive portion of a portion corresponding to the three-dimensional back surface of the bioactive center, and a linker such as a fishing line is bonded to the portion, and furthermore, the other end of the linker is attached to the other end of the linker. A substance having an affinity for a substance that cannot enter the receptor or a substance that cannot enter the receptor. The present invention has found that by performing such a chemical modification, the physiologically active center can be physiologically inactive while being preserved as it is. In addition, the present invention provides a novel tool capable of measuring the relationship between the affinity of a physiologically active substance for a receptor, the steric hindrance, and the affinity of a chemically modified moiety by such chemical modification.

The receptor of the present invention is not limited to a so-called receptor, but may be any receptor that forms pores such as a receptor (receptor), an ion channel, a transporter, and a pump. Ion channels and transporters such as sodium channel, calcium channel, amino acid transporter and serotonin transporter have pores penetrating the cell membrane and are preferred as the receptor of the present invention. The physiologically active substance of the present invention may be any substance that enters the receptor and expresses its physiological activity and that has a site that can be chemically modified on the back of the physiologically active center. If the molecular weight is too small, there is no chemically modifiable portion, which is not preferable. What is necessary is just to have a part which penetrates the interior of the receptor completely and is physiologically inactive. The physiologically active substance of the present invention may be any substance as long as it enters the receptor and acts to exert a physiological effect on cells, and may enhance or inhibit a physiological effect. It may be.
The substance may be a physiologically active substance in a living body, a substance such as a medicine, or a substance that becomes a toxic substance. Further, it may be a proteinaceous substance or a non-proteinaceous substance.

The linker of the present invention has a functional group capable of chemically bonding to a physiologically active substance or a chemically modified physiologically active substance at one end of a molecule, and a “substance that cannot enter a receptor” at the other end. Or a compound having a functional group capable of chemically bonding with a “substance having an affinity for a substance that cannot enter a receptor”, and a compound having a physiologically inactive portion between the functional groups at both ends. preferable. For example, those represented by the above-mentioned general formulas (I) and (II) or the linkers contained in the compounds obtained in Examples 5 and 6 described later are preferable. As a portion between the functional groups at both ends, in the case of a non-activated derivative,
For example, in the case of a derivative having 1 to 30 carbon atoms and a physiological activity, a group having a linear group having 20 to 50 carbon atoms may be used. In addition, some or all of the carbon atoms in the chain may be substituted with a hetero atom such as an oxygen atom, a nitrogen atom, or a sulfur atom, or may have a ring in the chain. Is also good. Such a ring may be a carbon ring, an oxygen atom, a nitrogen atom,
It may be a heterocyclic ring having a hetero atom such as a sulfur atom.

The substance which cannot enter the receptor of the present invention may be any molecule which is larger than the size of the entrance of the receptor and which does not enter the interior of the receptor. When the substance is directly bonded to a linker, it is a molecule having a functional group capable of chemically bonding to the linker molecule. Also,
When the substance does not directly bind to the linker, the substance has an affinity for the substance that directly binds to the linker. The substance can be labeled with a radioactive element, a fluorescent substance, or the like, if necessary.

The substance having an affinity for a substance which cannot enter the receptor of the present invention has an affinity for the above-mentioned "substance which cannot enter the receptor" and can be chemically bonded to a linker. Those having a functional group are preferred.
Although the affinity is not particularly limited, it may be a chemical affinity or a physical affinity, but one that can measure the affinity in an environment similar to that of a cell system is preferable.

The non-activated derivative of the physiologically active substance of the present invention has a physiologically active portion in the same form as a natural product and is physiologically inactivated. It is useful as an antigen. The antibody of the present invention can be produced according to a general antibody production method. Even if the physiologically active substance is a toxic substance, the non-activated derivative of the present invention can be administered to a sensitized substance as it is because it is detoxified. The non-activated derivative of the physiologically active substance of the present invention is administered to an animal such as a rat or a mouse to sensitize the cells, extract cells containing the antibody, and produce an antibody from the cells by a conventional method. For example, a cell fusion method or a method of cloning the antibody gene to obtain a chimeric antibody or a humanized antibody can be employed.

On the other hand, in the present invention, a portion of the physiologically active substance other than the receptor recognition site is chemically modified, a linker is bound to the chemically modified site, and the other end of the linker is immobilized on the carrier. Is, for example, polystyrene,
Examples include polypropylene, polyethylene, glass beads, silica gel, and polysaccharides (including derivatives). Also,
Examples of the substance having an affinity for the substance bound to the other end of the linker include avidin. Avidin itself can be used as a carrier. When avidin is used as a carrier or when avidin is immobilized on a carrier, the substance to be bound to the other end of the linker is biotin. The compound of the present invention having a sufficiently long linker (for example, Compound 6 described below) can be used to isolate a new sodium channel or calcium channel, for example, by using it with an avidin column. Also,
When the compound of the present invention (biotin analog), which has a long linker and retains its activity, is complexed with avidin to increase the molecular weight, the sensitivity increases. For example, binding on a sodium channel using a cryogenic electron microscope or the like is performed. The site can be clarified (the molecule of the poison itself is too small to be observed with an electron microscope, etc.). Therefore,
The compound of the present invention having a long linker and retaining the activity can be said to be an analog having high biological utility.

[0034]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these specific examples.

Example 1 Cys (MeBzl) 5-μ-
Manufacture of conotoxins Amino acids with removable protecting groups, Arg (Pmc), As
p (OtBu), Cys (Trt), Gln (Trt), Lys (Boc), Ser
Protected peptides having (t-Bu), Thr (tBu), Hyp (tBu) and MeBzlCys were prepared by solid phase synthesis using FMOC method. Protection peptide resin TFA- thioanisole _-H 2 O-phenol -EDT by under ice-cooling for 5 minutes, removed from the resin by stirring at room temperature for 1.5 hours, precipitated together with the resin with ether, filtered, 2
Elution with M acetic acid. The resin was washed with water, and this washing solution was added to the acetic acid solution, and the pH was adjusted to 7.8 with aqueous ammonia. This crude peptide (0.5 mM, 0.2 M ammonium acetate solution) was stirred for 1-2 days and air-oxidized. After lyophilization, Sephadex G-50F column with 30% acetic acid, ion exchange (0.01 M to 0.7 M ammonium acetate) chromatography with CM-cellulose CM-52,
And purified by ODS-HPLC (0.1% TFA-acetonitrile) and synthesized in 4.7% yield. The resulting HPLC and MALDI-TOF masses are shown in FIG.

Example 2 Production of Lys (Biot) 5-μ-conotoxin (Compound 2) Amino acid having a removable protecting group, Arg (Pmc), As
p (OtBu), Cys (Trt), Gln (Trt), Lys (Boc), Ser
(T-Bu), Thr (tBu), Hyp (tBu), and Lys (Bio
Using t), a protected peptide having Lys (Biot) at position 5 was prepared by a solid phase synthesizer using the FMOC method. Protection peptide resin TFA- thioanisole _-H 2 O-
Phenol-EDT for 5 minutes under ice cooling, room temperature 1.5
Removed from the resin by stirring for hours, precipitated with the resin in ether, filtered and eluted with 2M acetic acid.
The resin was washed with water, and this washing solution was added to the acetic acid solution, and the pH was adjusted to 7.8 with aqueous ammonia. This crude peptide (0.5 mM, 0.2 M ammonium acetate solution) was added to 1-
Stirred for 2 days and air oxidized. After lyophilization, Sephadex G-50F column with 30% acetic acid. Ion exchange (0.01M to 0.7M ammonium acetate) chromatography with CM-cellulose CM-52, and ODS-H
Purified by PLC (0.1% TFA-acetonitrile) and synthesized in 6.0% yield. The resulting HPLC and MALDI-TOF masses are shown in FIG.

Example 3 Production of S-Biotin PEAC5 Cystein-5-μ-conotoxin (Compound 3) (1) [Cys (MeBzl) 5] -μ-conotoxin was dissolved in HF and reacted at 0 ° C. for 1 hour. . After removing HF, it was dissolved in water, washed with ether, and freeze-dried.
The product was purified by ODS-HPLC (0.1% TFA-9% acetonitrile) to synthesize [Cys5] -μ-conotoxin in 65% yield. The resulting HPLC and MALDI-TOF masses are shown in FIG. (2) [C
[ys5] -μ-conotoxin was reacted with biotin PEAC5 maleimide in a 0.02 M ammonium acetate solution for 30 seconds, purified by gel filtration through Sephadex G25F (0.02 M ammonium acetate), and freeze-dried. The resulting MALDI-TOF mass is shown in FIG.

Example 4 Preparation of S-biotin PE5 Cystein-5-μ-conotoxin (Compound 4) [Cys5] -μ-conotoxin was reacted with biotin PE maleimide in 0.02 M ammonium acetate solution for 30 seconds to give Sephadex G25F Gel filtration (0.0
(2M ammonium acetate) and lyophilized. The resulting MALDI-TOF mass is shown in FIG.

Example 5 PE-EDT-BM (PEO)
₄ of [Cys5]-.mu.-conotoxins (Compound 5) (1) Preparation of biotin PE maleimide (3.4 mg) 0.0
Dissolve in 2 M ammonium acetate solution (pH 4.0, 1 ml) and add 0.02 μl of ethanedithiol (20 μl).
2M ammonium acetate solution (pH 4.0, 500 μl)
The mixture was added dropwise and stirred for 30 minutes. After the reaction, ethanedithiol was removed with diethyl ether (1 ml × 5), followed by freeze-drying. This was purified by RP-HPLC, freeze-dried again, and

[0040]

Embedded image

(PE-EDT) was obtained. FIG. 14 shows a mass spectrum chart of the obtained compound.
Shown in (2) In a 0.02 M ammonium acetate (pH 4.0) solution containing 5 mM BM (PEO) ₄ (20 μl),
A solution of [Cys5] -μ-conotoxin (0.1 μmol) dissolved in 0.02 M ammonium pH 4.0 (30 μl) was added, and the mixture was stirred for 10 minutes. To this was added 6 mM of the compound obtained in the above (1) (50 μl), and the mixture was stirred for 30 minutes. This solution was directly purified by RP-HPLC, freeze-dried, and

[0042]

Embedded image

The compound represented by the formula was obtained. FIG. 15 shows a mass spectrum chart of the obtained compound.

Example 6 PEAC-EDT-BM (PE
_{O) 4} of [Cys5]-.mu.-conotoxin (Compound 6)
Production of (1) Biotin PEAC5 maleimide (4.2 mg) in 0.02 M ammonium acetate solution (pH 4.0, 1 m
l) and dissolved in a 0.02 M ammonium acetate solution (pH 4.0, 50 μl) containing ethanedithiol (20 μl).
0 μl) and stirred for 30 minutes. After the reaction, ethanedithiol was removed with diethyl ether (1 ml × 5), followed by freeze-drying. This was purified by RP-HPLC, freeze-dried again, and

[0045]

Embedded image

(PEAC-EDT) was obtained. FIG. 1 shows a mass spectrum chart of the obtained compound.
6 is shown. (2) In a 0.02 M ammonium acetate solution (pH 4.0) containing 5 mM BM (PEO) ₄ (20 μl),
[Cys5] -μ-conotoxin (0.1 μmol) in 0.02 M ammonium acetate solution (pH 4.0, 30 μM)
The solution dissolved in l) was added and stirred for 10 minutes. To this was added 6 mM of the compound obtained in the above (1) (50 μl),
Stir for 30 minutes. This solution is directly used for RP-HPLC
And freeze-dried.

[0047]

Embedded image

The compound represented by the formula was obtained. FIG. 17 shows a mass spectrum chart of the obtained compound.

FIG. 18 is a photograph instead of a drawing showing the results of a skeletal muscle contraction experiment performed on compound 5 and compound 6. The vertical and horizontal axes in FIG. 18 are the same as those in FIG. The top row of FIG. 18 first shows PE-EDT-BM.
(PEO) ₄ μM [Cys5] -μ-conotoxin (compound 5) was added at 1 μM, then 3 μM, and when contraction of skeletal muscle was inhibited, avidin (Av) was added at 3 μM. Addition of avidin alleviates the inhibition of contraction. The second row from the top in FIG.
DT-BM _{(PEO) 4} of [Cys5]-.mu.-conotoxins (Compound 5) 1 [mu] M, then 3μM added, then shows the case of 3μM added streptavidin (Stav). It can be seen that the addition of streptavidin mitigated the inhibition of contraction as in the case of avidin. The third row from the top in FIG. 18 first shows PEAC-EDT-
BM _{(PEO) 4} of [Cys5]-.mu.-conotoxin (Compound 6) a 1 [mu] M, then 3μM added, streptavidin when the skeletal muscle contraction was inhibited (StA
v) shows the case where 3 μM was added. In this case, similarly to the top and second rows, the addition of streptavidin alleviates the inhibition of contraction. The bottom of FIG.
EAC-EDT-BM (PEO) ₄ [Cys5] -μ
-Shows a case where 1 μM of conotoxin (compound 6) was added and 1 μM of avidin (Av) was added when contraction of skeletal muscle was inhibited. In this case, even if avidin was added, inhibition of contraction was observed. It is understood that the activity is not relaxed, that is, the inhibitory activity of μ-conotoxin on skeletal muscle contraction is maintained as it is.

Test Example 1 Contraction test of rat skeletal muscle Rat diaphragm was excised, cut into a suitable size, and suspended in Ringer's solution. The tension generated by the electrical stimulation of this muscle specimen was measured electrically. Dissolve the sample in water,
In addition to this specimen, the resulting decrease in tension was measured. In addition, the process of recovering from the effect of the drug by changing the Ringer's solution or by further adding the drug was recorded.

Test Example 2 Measurement of Inhibitory Activity (IC ₅₀ ) on Skeletal Muscle Contraction [mu] -conotoxin GIIIA and μ-conotoxin GIIIA obtained by substituting cysteine for threonine at position 5
-With respect to various biotin analogs of conotoxin GIIIA, the degree of inhibitory activity on skeletal muscle contraction to electrical stimulation was measured. Table 1 shows the results.

[0052]

[Table 1]

[0053]

Industrial Applicability The present invention chemically modifies a physiologically inactive part of a physiologically active substance, preferably a part sterically behind the physiologically active part, and binds a linker part and a sterically large molecule to this. By doing so, it is possible to provide a physiologically inactive derivative retaining the physiologically active portion as it is and a derivative retaining the physiological activity. The present invention also provides a novel method for deactivating a physiologically active substance and an antibody against a non-activated derivative of the physiologically active substance.
Since the non-activated derivative of the present invention retains the physiologically active center as it is, not only is it easy to produce antibodies even if it is a toxic substance, but also to analyze the structure of the receptor for the physiologically active substance. It also provides a new method.
In addition, by using the compound of the present invention having a long linker and retaining the activity, it is possible to isolate a new sodium channel or calcium channel. Furthermore, by using the compound of the present invention having a long linker and retaining the activity, the binding site on the sodium channel can be clarified using, for example, a cryogenic electron microscope.

[Brief description of the drawings]

FIG. 1 schematically shows the relationship between a physiologically active substance and a receptor.

FIG. 2 schematically shows the relationship between a non-activated derivative of the present invention and a receptor.

FIG. 3 schematically shows the relationship between a non-activated derivative and a receptor according to another embodiment of the present invention.

FIG. 4 schematically shows the amino acid sequence of μ-conotoxin GIIIA and its three-dimensional structure.

FIG. 5 shows the IC ₅₀ when the amino acid at each position of μ-conotoxin GIIIA was substituted with alanine or lysine.
Is a graph. Black indicates the case where alanine was substituted, gray indicates the case where lysine was substituted, and NT indicates the case where the test was not performed.

FIG. 6 shows μ-conotoxin GIIIA (μGIII
A) (upper part of FIG. 6) μ-conotoxin GIIIA in which threonine at position 5 has been replaced with S-methylbenzylcysteine
(Cys (MeBzl) 5) (middle of FIG. 6) and μ-conotoxin G in which the 5-position is substituted with N-biotinylated lysine
6A is a photograph instead of a drawing showing the result of a skeletal muscle contraction experiment on rat skeletal muscle for IIIA (Lys (Biot) 5) (lower part of FIG. 6). The vertical axis in FIG. 6 is the skeletal muscle tension (mN
(Millinewton)), and the horizontal axis in FIG. 6 is time (minute).
It is.

FIG. 7. Lys (Biot) 5 and avidin (A).
When v) is added at the same time (the top row in FIG. 7), when avidin (Av) is added first, and then when Lys (Biot) 5 is added (the second row from the top in FIG. 7), Lys (Bio) is first added.
t) When 5 was added and then avidin was added to the system (third row from the top in FIG. 7), μ-conotoxin GIII
7A is a photograph instead of a drawing, showing the results of a skeletal muscle contraction experiment on rat skeletal muscle in the case of only A (μGIIIA) (the bottom row of FIG. 7). The vertical axis in FIG. 7 is the skeletal muscle tension (mN
(Millinewton)), and the horizontal axis in FIG. 7 is time (minute).
It is.

FIG. 8 shows first the non-activated derivative A (biotin PE
AC5 maleimide) and then avidin (Av)
Is added (the top row of FIG. 8), the non-activated derivative A
(Biotin PEAC5 maleimide) and avidin are added simultaneously (the second row from the top in FIG. 8), and non-activated derivative B (biotin PE maleimide) is added followed by avidin (Av) (FIG. 8 (third row from the top) is a photograph replacing a drawing, showing the results of a skeletal muscle contraction experiment of rat skeletal muscle. The vertical axis in FIG. 8 indicates the skeletal muscle tension (mN (millinewton)), and the horizontal axis in FIG. 8 indicates time (minute).

FIG. 9 shows a chart of HPLC (left) and MALDI-TOF mass (right) of [Cys (MeBzl) 5] -μ-conotoxin.

FIG. 10 shows [Lys (Biot) 5] -μ-
The charts of conotoxin HPLC (left) and MALDI-TOF mass (right) are shown.

FIG. 11 shows a chart of [Cys5] -μ-conotoxin HPLC (left) and MALDI-TOF mass (right).

FIG. 12 shows MALDI-TOF of biotin PEAC5 maleimidated [Cys5] -μ-conotoxin.
3 shows a chart of cells.

FIG. 13 shows biotin PE maleimide [Cys
5 shows a chart of the MALDI-TOF mass of 5] -μ-conotoxin.

FIG. 14 shows a mass spectrum chart of PE-EDT.

FIG. 15 shows PE-EDT-BM (PEO) ₄
1 shows a mass spectrum chart of [Cys5] -μ-conotoxin.

FIG. 16 shows a mass spectrum chart of PEAC-EDT.

FIG. 17 is a diagram showing a PEAC-EDT-BM (PE
_{O) 4} of [Cys5]-.mu.-shows the conotoxin mass spectrum chart.

FIG. 18 is a diagram showing a PE-EDT-BM (PE
_{O) 4} of [Cys5]-.mu.-conotoxin (Compound 5)
, And then avidin (Av) (the top row in FIG. 18), first, PE-EDT-BM (PEO) ₄
[Cys5] -μ-conotoxin (compound 5) and then streptavidin (StAv) (second stage from the top in FIG. 18), first PEAC-EDT
-BM (PEO) ₄ [Cys5] -μ-conotoxin (compound 6) is added, followed by streptavidin (S
tAv) When avidin (Av) was added (third stage from the top in FIG. 18), and PEAC-EDT-BM (P
_{EO) 4} of added [Cys5]-.mu.-conotoxin (Compound 6), followed by the avidin (when added Av) (bottom of FIG. 18), illustrates the results of a skeletal muscle contraction experiments in rat skeletal muscle This is an alternative photo. The vertical axis of FIG. 8 indicates the skeletal muscle tension (mN (millinewton)),
The horizontal axis in FIG. 8 is time (minute).

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Receptor 2 Cell membrane 3 Receptor action site 4 Physiologically active substance 5 Receptor recognition site of bioactive substance 6 Linker 7 Substance which cannot enter receptor 8 Substance which cannot enter receptor Substance

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/18 C07K 16/18 17/02 17/02 C12P 21/08 C12P 21/08 G01N 33/53 G01N 33/53 D 33/566 33/566 33/68 33/68

Claims (55)

[Claims] 1. A substance which expresses a biological activity by binding to an internal action site of a receptor, wherein a linker is bound to a portion other than the receptor recognition site of the physiologically active substance, and the receptor is attached to the other end of the linker. A substance that cannot enter the body or a substance that has an affinity with a substance that cannot enter the receptor. The bioactive substance is inactive while the receptor recognition site of the physiologically active substance is preserved. How to 2. The method according to claim 1, wherein a portion other than the receptor recognition site of the physiologically active substance is chemically modified, and a linker is bound to the chemically modified site. 3. The method according to claim 1, wherein the portion other than the receptor recognition site of the physiologically active substance is three-dimensionally a back portion of the receptor recognition site. 4. The method according to claim 1, wherein the substance that binds to one end of the linker is a substance that cannot enter a receptor. 5. The method according to claim 1, wherein the substance that binds to one end of the linker has a affinity for a substance that cannot enter a receptor. 6. The method according to claim 1, wherein the physiologically active substance is a protein. 7. The bioactive substance is a toxic substance.
The method according to any of the above. 8. The method according to claim 1, wherein the receptor is an ion channel. 9. The method according to claim 8, wherein the ion channel is a sodium channel or a calcium channel. 10. The method according to claim 7, wherein the physiologically active substance is μ-conotoxin. 11. A substance which expresses a biological activity by binding to an internal action site of a receptor, and chemically modifies a portion of the physiologically active substance other than the receptor recognition site, and binds a linker to the chemical modification site. And a non-activated derivative of a physiologically active substance comprising a substance that cannot enter the receptor or a substance that has an affinity for a substance that cannot enter the receptor. 12. The non-activated derivative according to claim 11, wherein the portion other than the receptor recognition site of the physiologically active substance is sterically a back portion of the receptor recognition site. 13. The non-activated derivative according to claim 11, wherein the linker comprises a compound having 1 to 30 carbon atoms which may have hetero atoms having functional groups at both ends. 14. The non-activated derivative according to claim 11, wherein the substance that cannot enter the receptor is a high molecular substance. 15. The non-activated derivative according to claim 14, wherein the polymer substance is a protein. 16. The method according to claim 1, wherein the protein is avidin.
6. The non-activated derivative according to 5. 17. The substance having an affinity for a substance that cannot enter a receptor is biotin.
7. The non-activated derivative according to any of 6. 18. The non-activated derivative according to claim 11, wherein the physiologically active substance is a peptide or a protein. 19. The biologically active substance is a toxic substance.
2. The non-activated derivative according to 1.). 20. The non-activated derivative according to claim 18 or 19, wherein the chemical modification is substitution with an amino acid having a functional group. 21. The non-activated derivative according to any one of claims 11 to 20, wherein the chemical modification is for easily forming a bond with a linker. 22. The non-activated derivative according to claim 11, wherein the receptor is an ion channel. 23. The non-activated derivative according to claim 22, wherein the ion channel is a sodium channel or a calcium channel. 24. The non-activated derivative according to claim 19, wherein the physiologically active substance is μ-conotoxin. 25. The non-activated derivative according to claim 24, wherein the chemical modification is substitution of threonine at position 5 with cysteine. 26. A non-activated antigen comprising a non-activated derivative according to any one of claims 11 to 25, wherein the receptor recognition site of a physiologically active substance is conserved. 27. The bioactive substance is a toxic substance.
The antigen according to 1. 28. A method for producing an antibody against the inactivated antigen according to claim 26 or 27 by sensitizing and immunizing an animal. 29. An antibody against the non-activated antigen according to claim 26 or 27. 30. The antibody according to claim 29, wherein the antibody is a monoclonal antibody or a polyclonal antibody. 31. Inactivation of a physiologically active substance according to any one of claims 11 to 25, wherein a substance having an affinity for a substance which cannot enter a receptor is bound to one end of the cell. Treatment with a derivative to render the receptor-recognition site of the biologically active substance capable of binding to the receptor, and then treatment of the cell line with a substance that cannot enter the receptor to reduce the activity of the receptor. A method for measuring the affinity between the receptor and a physiologically active substance. 32. The method according to claim 31, wherein the measurement is performed while changing the length of the linker. 33. The method according to claim 31, wherein the substance having an affinity for the substance that cannot enter the receptor is biotin, and the substance that cannot enter the receptor is avidin. 34. The cell, wherein a substance which cannot enter a receptor is bound to one end of the linker.
25. Measure the distance between the entrance of the receptor and the site of action of the physiologically active substance by treating with the non-activated derivative of the physiologically active substance according to any one of 25. Method. 35. The method according to claim 34, wherein the measurement is performed while changing the length of the linker. 36. The cell according to any one of claims 11 to 25, wherein a substance corresponding to the size of the entrance of the receptor is bound to one end of the linker having a length not reaching the site of action of the receptor. A method for measuring the size of the entrance of the receptor, which comprises treating the receptor with a non-activated derivative of a physiologically active substance and measuring the activity of the receptor. 37. The method according to claim 36, wherein the measurement is performed while changing the steric size of the substance bound to the linker. 38. In a substance that expresses a physiological activity by binding to an internal action site of a receptor, a portion other than the receptor recognition site of the physiologically active substance is chemically modified, and a linker is bound to the chemically modified site. Then, the other end of the linker is fixed to a carrier, or a substance that can be fixed to the carrier (or has an affinity with the carrier) or a substance that has an affinity for the substance fixed to the carrier is fixed to the other end of the linker. A derivative of a physiologically active substance that retains its biological activity and is bound. 39. The derivative according to claim 38, wherein the portion other than the receptor recognition site of the physiologically active substance is sterically a back portion of the receptor recognition site. 40. The derivative according to claim 38, wherein the linker comprises a compound having 30 to 50 carbon atoms which may have hetero atoms having functional groups at both ends. 41. The derivative according to claim 38, wherein the carrier is polystyrene, polypropylene, polyethylene, glass beads, silica gel, or a polysaccharide (including a derivative). 42. The derivative according to claim 38, wherein the substance fixed on the carrier is a polymer substance. 43. The derivative according to claim 42, wherein the polymer substance is a protein. 44. The protein of claim 4, wherein the protein is avidin.
3. The derivative according to 3. 45. The derivative according to claim 38, wherein the substance having an affinity for the substance fixed on the carrier is biotin. 46. The derivative according to claim 38, wherein the carrier is avidin, and the substance having an affinity for the carrier is biotin. 47. The derivative according to any one of claims 38 to 46, wherein the physiologically active substance is a peptide or a protein. 48. The bioactive substance is a toxic substance.
The derivative according to 1. 49. The derivative according to claim 47, wherein the chemical modification is substitution with an amino acid having a functional group. 50. The derivative according to any one of claims 38 to 49, wherein the chemical modification is for easily forming a bond with a linker. 51. The derivative according to any one of claims 38 to 50, wherein the receptor is an ion channel. 52. The derivative according to claim 51, wherein the ion channel is a sodium channel or a calcium channel. 53. The derivative according to claim 48, wherein the physiologically active substance is μ-conotoxin. 54. The derivative according to claim 53, wherein the chemical modification is a substitution of threonine at position 5 with cysteine. 55. A method for isolating a sodium channel or a calcium channel using the derivative according to any one of claims 38 to 54.