JP6622079B2 - Macrocyclic molecule and screening method thereof - Google Patents

Description

  The present invention relates to a macrocyclic molecule having a cryptand structure including a peptide moiety and a crown ether moiety, and a method for identifying the macrocyclic molecule. In particular, the present invention relates to macrocycle molecules that bind to target molecules, such as target proteins, and methods for identifying such macrocycle molecules by screening.

  Artificial macrocycle molecules are attracting attention because of their broad applicability, such as electrochemical materials and photophysical materials. Among the artificial macrocycle molecules, crown ethers and analogs thereof (crown analogs) have excellent biocompatibility because their hydrophilic oligoethylene structure increases water solubility and prevents aggregation.

  Despite the potential usefulness of the crown, no comprehensive studies on its biological application have been reported until recently. The present inventors have reported for the first time the construction of a crown ether-like molecular library (crown library) that provides a very large molecular diversity of 10 9 using T7 phage. A molecule that specifically binds to a cancer-related protein (heat shock protein 90; Hsp90) was successfully identified and isolated (Patent Document 1, Non-Patent Document 1).

JP 2014-128211 A

Chem. Comm. (2014) Vol. 50, No. 30, pp. 3887-4012

However, there is a need for obtaining artificial macrocycle molecules with more diverse structures. In particular, a crown ether-like molecule obtained from a prior-art crown library has a binding affinity (dissociation constant K _D ) of about 1.7 μM for a target protein, and exhibits a binding force equivalent to or stronger than that. There is a need for obtaining molecules.

  An object of the present invention is to provide a novel macrocyclic molecule. In particular, it is an object to provide a macrocyclic molecule that can bind to a protein. In particular, it is an object of the present invention to provide a macrocyclic molecule capable of binding to Hsp90, and a peptide composition for producing such a macrocyclic molecule. It is another object of the present invention to provide a library and a screening method that enable identification and isolation of macrocyclic molecules that can bind to a target molecule.

The present invention includes the following aspects.
[1]
A molecule having a structure represented by the following formula (I):
Where
X ¹ to X ¹⁴ (including ^{C 4} and ^{C 12)} represents amino acids joined by peptide bonds, ^{C 4} and ^{C 12} each represents a cysteine, ^{S *} is derived from cysteine ^{C 4} and ^{C 12} Represents a sulfur atom,
X ¹ is any amino acid,
X ² is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ³ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁵ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁶ is any amino acid,
X ⁷ is any amino acid,
X ⁸ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁹ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ¹⁰ is any amino acid,
X ¹¹ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ¹³ is any amino acid,
X ¹⁴ is any amino acid or absent,
R ¹ is a group having a structure represented by any of the following formulas (II) to (V):
Where
n is an integer of 1 to 4,
R ^{2 to} R ⁵ are each independently hydrogen or a methyl group.
molecule.
[2]
X ² is tryptophan,
X ³ is valine,
X ⁵ is leucine,
X ⁸ is tryptophan,
X ⁹ is leucine,
X ¹¹ is isoleucine,
The molecule according to [1].
[3]
The molecule according to [1] or [2], wherein X ⁷ is proline.
[4]
X ¹ , X ⁶ , X ¹⁰ , and X ¹³ are each independently a hydrophilic amino acid selected from the group consisting of glutamine, asparagine, serine, threonine, arginine, lysine, histidine, tyrosine, aspartic acid, and glutamic acid. The molecule according to any one of [1] to [3].
[5]
X ¹ is glutamine or asparagine,
X ⁶ is glutamine or asparagine,
X ¹⁰ is serine or threonine;
X ¹³ is arginine or lysine,
The molecule according to any one of [1] to [4].
[6]
The peptide chain portion represented by X ^{1 to} X ¹⁴ (including C ⁴ and C ¹² ) is
QWVCLNPWLSICRA
Having an amino acid sequence of
The molecule according to any one of [1] to [5].
[7]
The molecule according to any one of [1] to [6], wherein in R ¹ , n = 2.
[8]
R ¹ is a group having a structure represented by the following formula (II):
Where n = 2.
The molecule according to any one of [1] to [7].
[9]
A molecule having a structure represented by the following formula (VI).
[10]
The following sequence ^{X 1 -X 2 -X 3 -C 4} -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C 12 -X 13 -X 14
A composition for producing an hsp90 binding agent comprising a peptide having:
X ^{1 to} X ¹⁴ (including C ⁴ and C ¹² ) represent amino acids linked by peptide bonds, C ⁴ and C ¹² each represent cysteine,
X ¹ is any amino acid,
X ² is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ³ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁵ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁶ is any amino acid,
X ⁷ is any amino acid,
X ⁸ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁹ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ¹⁰ is any amino acid,
X ¹¹ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ¹³ is any amino acid,
X ¹⁴ is any amino acid or absent,
The peptide is linked to the two nitrogen atoms of the azacrown ether via the C ⁴ and C ¹² cysteine side chains to yield an hsp90 binding agent,
Composition.
[11]
X ² is tryptophan,
X ³ is valine,
X ⁵ is leucine,
X ⁸ is tryptophan,
X ⁹ is leucine,
X ¹¹ is isoleucine,
[10] The composition according to [10].
[12]
The composition according to [10] or [11], wherein X ⁷ is proline.
[13]
X ¹ , X ⁶ , X ¹⁰ , and X ¹³ are each independently a hydrophilic amino acid selected from the group consisting of glutamine, asparagine, serine, threonine, arginine, lysine, histidine, tyrosine, aspartic acid, and glutamic acid. The composition according to any one of [10] to [12].
[14]
X ¹ is glutamine or asparagine,
X ⁶ is glutamine or asparagine,
X ¹⁰ is serine or threonine;
X ¹³ is arginine or lysine,
[10] The composition according to any one of [13].
[15]
The peptide chain portion represented by X ^{1 to} X ¹⁴ (including C ⁴ and C ¹² ) is
QWVCLNPWLSICRA
Having an amino acid sequence of
[10] The composition according to any one of [14].
[16]
T7 phage in which the peptide part of the molecule according to any one of [1] to [9] is fused to the C-terminus of the gp10 protein.
[17]
A method of screening for molecules that can bind to a target molecule,
(A) genetically engineered so that a peptide containing two cysteines separated by 4-10 random amino acids is fused to the C-terminus of the gp10 protein; and
Live of modified T7 phage comprising a structure in which —CO—CH ₂ — groups linked to two opposite nitrogen atoms on the periphery of azacrown ether are respectively bonded to sulfur atoms of side chains of the two cysteines Rally,
Mixing with the target molecule;
(B) separating the modified T7 phage bound to the target molecule;
(C) infecting a host bacterial cell with the isolated modified T7 phage, and propagating the corresponding T7 phage;
(D) reacting the T7 phage grown in the step (c) with a dihalogen compound having a structure in which a haloacetyl group or a haloacetamide group is linked to the two nitrogen atoms of the azacrown ether, Obtaining a new phage;
(E) mixing the modified T7 phage newly obtained in the step (d) with the target molecule, and (f) separating the modified T7 phage bound to the target molecule,
Including a method.
[18]
After step (f)
(G) The method according to [17], further comprising a step of repeating the steps (c) to (f).
[19]
The method according to [18], wherein in the step (g), the steps (c) to (f) are repeated at least four times.
[20]
Prior to step (a), the method further comprises an initial step of creating a library of the modified T7 phage,
A library of T7 phage genetically engineered such that a peptide containing two cysteines separated by 4-10 random amino acids is fused to the C-terminus of the gp10 protein,
The method according to any one of [17] to [19], comprising reacting with a dihalogen compound having a structure in which a haloacetyl group or a haloacetamide group is linked to the two nitrogen atoms of the azacrown ether.
[21]
A gene engineered such that a peptide containing two cysteines separated by 4-10 random amino acids is fused to the C-terminus of the gp10 protein; and
Live of modified T7 phage comprising a structure in which —CO—CH ₂ — groups linked to two opposite nitrogen atoms on the circumference of azacrown ether are respectively bonded to sulfur atoms of the side chains of the two cysteines Rally.

According to the present invention, macrocycle molecules having various binding forces with respect to a target molecule can be obtained efficiently. In particular, it is possible to obtain such can become an alternative to antibodies, strong binding molecules having a K _D of nM range.

FIG. 1 shows (A) of 1,1 ′-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl) bis (2-bromoethanone) synthesized according to Scheme 1. ) LC-MS spectrum (actual m / z values = 503.3, 505.2, 507.2; theoretical m / z values = 503.0, 505.0, 507.0) and (B) ¹ H NMR spectrum (500 MHz, CDCl ₃ ). FIG. 2 shows the result of analyzing a model peptide modified with crown ether by LC-MS / MS. FIG. 3 shows the intensity of fluorescence observed when (A) the model peptide was modified with different concentrations of crown ether and then with FL-IA (fluorescein iodoacetamide), and (B) the fluorescence intensity at A. A quantified graph is shown. FIG. 4 shows an overview of the screening according to one embodiment of the present invention. FIG. 5 shows the results of quantification of binding to target protein or control protein (BSA) by ELISA for phage groups (polyclones) after each round of positive selection. FIG. 6 shows the results of quantifying binding to target protein or control protein (BSA) by ELISA for 22 phage clones isolated after screening. FIG. 7 shows an LC-MS / MS spectrum of a cryptand molecule produced using a synthetic peptide according to one embodiment of the present invention. FIG. 8 shows the CD spectra of the synthetic peptide linker (top) and the corresponding cryptand molecule (bottom). FIG. 9 shows the results of an ITC experiment in which GST-Hsp90NTD (left) or GST (right) was dropped onto a cryptand molecule according to an embodiment of the present invention. N: number of binding sites, K _D : dissociation constant, ΔH: enthalpy change, ΔS: entropy change, ΔG: free energy change. FIG. 10 shows the ¹ H NMR spectrum (upper) of the cryptand molecule according to one embodiment of the present invention and the STD-NMR measurement result (lower) when bound to the target protein. On the upper left, the structure of this cryptand molecule and the arrangement of hydrophobic amino acids are shown.

  The first aspect of the invention provides a molecule capable of binding to Hsp90 protein. Hsp90 is an evolutionarily conserved protein that functions as a so-called molecular chaperone that controls protein quality in cells, as is known to those skilled in the art. Hsp90 plays an important role in various biological functions such as protein homeostasis, cell signaling and stress response. Hsp90 is also known to be involved in cancer pathology and is considered an important target in drug therapy (J. Med. Chem., 2010, 53, 3-17; Nat. Rev. Cancer, 2010, 10, 537-549). Therefore, molecules that bind to Hsp90 can be useful as components of Hsp90 binding agents, for example, as pharmaceutical components, or as research tools for performing inhibition, detection, purification, etc. based on binding.

The present inventors have previously developed a method (10BASE _d -T) in which a compound is selectively thioether bonded to a cysteine-containing peptide fused to the C-terminus of the gp10 protein of T7 phage (Patent Document 1, Non-Patent Document). 1).

In the present invention, using the 10BASE _d- T method, the inventors of a cryptand molecule having a structure in which a crown ether is linked to a random peptide (that is, a peptide containing a random amino acid other than two cysteines). When a library was constructed and molecules that bind to Hsp90 were screened, cryptand molecules that bind to Hsp90 with strong affinity were successfully identified and obtained.

  As used herein, the term “cryptand” generally encompasses crown ether-like cyclic molecules that take the form of three or more arcs attached. The molecule according to one embodiment of the present invention is a cryptand because a peptide arc is joined in such a way as to bisect the crown ether ring, resulting in a cyclic molecule containing three arcs.

In the present specification, the term “crown ether” includes an azacrown ether in which nitrogen is arranged in place of the oxygen of the ether. In fact, the cryptands of embodiments of the present invention comprise a first oxygen atom on the circumference of the crown and the furthest away from the first oxygen atom on the circumference of the crown (ie, the first oxygen atom) An azacrown ether in which a second oxygen atom (opposite) is replaced by a nitrogen atom, or an azacrown ether in which other oxygen on the circumference of the crown is also nitrogen. Opposed on the peripheral crown these two nitrogen atoms, -CO-CH ₂ - linker containing group (e.g., _{-CO-CH 2 -, - CH} 2 -CH 2 -NH-CO-CH 2 - , etc.) Are linked to the side chain sulfur atoms of the two cysteines of the above peptide, respectively (see, for example, the following formulas (I) to (V)).

  It has been found that the molecule can bind to the Hsp90 protein with a high affinity comparable to antibodies. This is a strong binding force far exceeding that of the prior art crown analog (Non-Patent Document 1).

The molecule according to one embodiment of the present invention belongs to the structure represented by the following general formula (I).
In this formula, X ^{1 to} X ¹⁴ (including C ⁴ and C ¹² ) represent amino acids linked by peptide bonds, C ⁴ and C ¹² each represent cysteine, and S ^* represents C ⁴ and C ¹² It represents a sulfur atom derived from the side chain of cysteine, and R ¹ represents a group containing a crown ether structure. In the portion consisting of a plurality of amino acids linked by the peptide bond, that is, the peptide portion, X ¹ is on the N-terminal side and X ¹⁴ is on the C-terminal side.

  By physicochemical analysis of the molecules according to embodiments of the present invention, the peptide moiety in the cryptand provides an arrangement of hydrophobic amino acids that appear to play a major role in binding to Hsp90, and the crown ether moiety improves water solubility. In addition to contributing to hydrogen bonds, it is suggested that moderate rigidity is given to the whole molecule, and that the strong bonding force described above is obtained as a result of these characteristics.

That is, the molecule according to one embodiment of the present invention includes a structure represented by the above general formula (I),
X ¹ is any amino acid,
X ² is a hydrophobic amino acid,
X ³ is a hydrophobic amino acid,
X ⁵ is a hydrophobic amino acid,
X ⁶ is any amino acid,
X ⁷ is any amino acid,
X ⁸ is a hydrophobic amino acid,
X ⁹ is a hydrophobic amino acid,
X ¹⁰ is any amino acid,
X ¹¹ is a hydrophobic amino acid,
X ¹³ is any amino acid,
X ¹⁴ is any amino acid, or X ¹⁴ is absent and X ¹³ is the terminal amino acid.

X ^{1 to} X ¹⁴ (excluding C ⁴ and C ¹² ) are preferably not cysteine.

  The hydrophobic amino acid is preferably selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine and methionine, more preferably selected from the group consisting of leucine, isoleucine, valine, tryptophan and phenylalanine, Preferably, it is selected from the group consisting of leucine, isoleucine, valine, and tryptophan. These hydrophobic amino acids are involved in hydrophobic bonds between peptides, and it is known to those skilled in the art that these hydrophobic amino acids are often replaced by other of these hydrophobic amino acids during evolution. Yes. That is, it is understood that these hydrophobic amino acids have a certain compatibility.

In the general formula (I), it is particularly preferable that X ² is tryptophan, X ³ is valine, X ⁵ is leucine, X ⁸ is tryptophan, X ⁹ is leucine, and X ¹¹ is isoleucine.

In general formula (I), X ⁷ is an arbitrary amino acid, but is preferably proline.

In general formula (I), X ¹ , X ⁶ , X ¹⁰ , and X ¹³ are arbitrary amino acids, but are preferably hydrophilic amino acids. This is because the presence of these hydrophilic amino acids is thought to further complement the water solubility and / or hydrogen bonding provided by the oxygen or nitrogen atoms of the crown ether moiety.

  The hydrophilic amino acid is preferably selected from the group consisting of glutamine, asparagine, serine, threonine, arginine, lysine, histidine, tyrosine, aspartic acid, and glutamic acid, more preferably glutamine, asparagine, serine, threonine, arginine. , Lysine, and histidine, and more preferably selected from the group consisting of glutamine, asparagine, serine, threonine, arginine, and lysine.

In general formula (I), it is more preferable that X ¹ is glutamine or asparagine, X ⁶ is glutamine or asparagine, X ¹⁰ is serine or threonine, and X ¹³ is arginine or lysine. It is particularly preferred that X ¹ is glutamine, X ⁶ is asparagine, X ¹⁰ is serine, and X ¹³ is arginine.

  In the molecule according to the embodiment of the present invention, it is particularly preferred that the peptide chain portion has an amino acid sequence of QWVCLNPWLSICRA. These alphabets represent amino acids by one-letter abbreviations commonly used by those skilled in the art.

The moiety represented by R ¹ in the above general formula (I) is a group containing an azacrown ether structure, and this azacrown ether is located at one nitrogen on the circumference of the crown and at an opposite position on the circumference of the crown. It is linked to the rest of the structure of general formula (I) via some other nitrogen. Non-carbon atoms other than these two nitrogens on the circumference of the crown may be oxygen or nitrogen. The crown size can be from 12-crown-4 to 30-crown-10, with 18-crown-6 being particularly preferred.

Preferably, R ¹ has a structure represented by any of the following formulas (II) to (V).

  In each of the formulas (II) to (V), the nitrogen atoms shown at the left and right ends are covalently bonded to the carbonyl carbon shown in the general formula (I).

In formula (II)-(V), n is an integer of 1-4. R ^{2 to} R ⁵ may each independently be hydrogen, a hydrocarbon group having 12 or less carbon atoms, or a hydrocarbon group having 6 or less carbon atoms. Examples of the case where R ^{2 to} R ⁵ are hydrocarbon groups include substituted or unsubstituted linear, branched, or cyclic alkyl groups or aryl groups, or composite groups thereof. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, sec-butyl, cyclohexyl, phenyl and the like, and methyl is particularly preferable.

In the general formula (I), the R ¹ group is preferably a group represented by the formula (II). In each of the formulas (II) to (V), it is preferable that n = 2.

A preferred example of the molecule of the present invention is one having a structure represented by the following formula (VI).

  The compound represented by the general formula (I) may form a part of a larger molecule. For example, the peptide moiety may be linked to additional amino acids on the N-terminal side and / or C-terminal side. In particular, the N-terminus of the peptide represented by the general formula (I) may be fused to the C-terminus of the gp10 protein of T7 phage. That is, the molecule according to one embodiment of the present invention may consist of the structure represented by the general formula (I) and the gp10 polypeptide moiety. Molecules according to embodiments of the present invention may include detection / purification tags or other oligopeptides or polypeptides instead of or in addition to gp10.

Crown ethers and cryptands are known to have the property of trapping metal ions in their rings. A cryptand molecule according to an embodiment of the present invention can be provided in the form of a complex in which one metal ion is incorporated in the ring by mixing with the metal ion. Examples of preferred metal ions in this case include, but are not limited to, Mg ²⁺ , Fe ²⁺ , Co ²⁺ , Ni ²⁺ , Cu ²⁺ , and Zn ²⁺ .

  The cryptand molecule according to the embodiment of the present invention can be obtained through a step of modifying the display peptide of T7 phage with crown ether, as will be described in detail below. Alternatively, it can also be obtained by synthesizing a peptide part or expressing it in bacterial cells or eukaryotic cells and purifying it, and then linking a crown ether part. These experimental techniques are within the ordinary skill of those skilled in the art.

In another aspect of the invention,
^{X 1 -X 2 -X 3 -C 4} -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C 12 -X 13 -X 14
A composition for producing an hsp90 binding agent is provided, which comprises a peptide having the amino acid sequence represented by: The peptides in the composition, via the side chain of a cysteine of C ⁴ and C ^12, are connected to two opposite nitrogen atoms on the crown lap aza crown ether, applications that cause hsp90 binder Have The types of amino acids X ^{1 to} X ¹⁴ are the same as those described above in relation to the specific cryptand molecule.

That is, in the above peptide-containing composition according to an embodiment of the present invention, X ¹ is an arbitrary amino acid, and X ² is a hydrophobicity selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine. X ³ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine, and X ⁵ is from leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine. A hydrophobic amino acid selected from the group consisting of: X ⁶ is any amino acid, X ⁷ is any amino acid, and X ⁸ is from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine. A selected hydrophobic amino acid, X ⁹ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine, X ¹⁰ is any amino acid, and X ¹¹ is , Leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine, a hydrophobic amino acid, X ¹³ is any amino acid, X ¹⁴ is any amino acid, or X ¹⁴ is present do not do. X ^{1 to} X ¹⁴ (excluding C ⁴ and C ¹² ) are preferably not cysteine.

In a peptide-containing composition according to an embodiment of the present invention, X ² is tryptophan, X ³ is valine, X ⁵ is leucine, X ⁸ is tryptophan, X ⁹ is leucine, and X ¹¹ is Isoleucine is preferred. X ⁷ is preferably proline.

In the peptide-containing composition according to an embodiment of the present invention, X ¹ , X ⁶ , X ¹⁰ , and X ¹³ are each independently glutamine, asparagine, serine, threonine, arginine, lysine, histidine, tyrosine, aspartic acid, And a hydrophilic amino acid selected from the group consisting of glutamic acid. More preferably, X ¹ is glutamine or asparagine, X ⁶ is glutamine or asparagine, X ¹⁰ is serine or threonine, and X ¹³ is arginine or lysine. It is particularly preferred that X ¹ is glutamine, X ⁶ is asparagine, X ¹⁰ is serine, and X ¹³ is arginine.

The peptide in the peptide-containing composition according to an embodiment of the present invention comprises:
QWVCLNPWLSICRA
Most preferably, it has the amino acid sequence

Components other than peptides may be contained in the composition, and examples of such components include water, reducing agents (particularly those used in 10BASE _d- T), buffers, salts (for example, chloride). Sodium, potassium chloride and the like), preservatives, and any combination thereof.

  Another aspect of the invention relates to a T7 phage containing a gp10 protein fused to the peptide portion of a cryptand molecule according to an embodiment of the invention described above. As used herein, “fusion” means that two or more peptides are linked by a peptide bond to form one peptide. As used herein, “peptide” includes oligopeptides and polypeptides.

  The C-terminus of gp10 and the N-terminus of the peptide part of the molecule of formula (I) may be fused by direct peptide bonding or may be fused via a linker amino acid. A linker amino acid is one or more amino acids inserted between the sequence of gp10 and the sequence of the peptide shown in formula (I). The linker amino acid may include an enzyme recognition sequence for enabling cleavage by a cleavage enzyme known to those skilled in the art, a tag sequence used for detection or purification, and the like. Since the modified peptide portion is cleaved by cleaving with a cleaving enzyme, a cryptand molecule containing no gp10 portion according to the first aspect of the present invention can be obtained.

The T7 phage according to the embodiment of the present invention can be propagated by infecting host bacterial cells in the same manner as normal T7 phage. However, what occurs during growth is a phage having a naked (unmodified) peptide chain from which the crown ether modification has been removed. The phages grown in this way can be obtained in large quantities with the same structure as the crown ether-modified T7 phage before growth by religating the crown ether structure using the 10BASE _d- T method. .

  A novel molecule that binds to a target protein other than Hsp90 or other target molecules by changing the amino acid sequence of the peptide portion while taking advantage of the advantages provided by the crown ether portion (or the advantage based on the cryptand structure) It is understood that it can be searched and acquired. It is also possible to search for additional Hsp90 binding molecules that exhibit different binding forces. Thus, another aspect of the invention relates to a method for screening for cryptand molecules that can bind to a target molecule.

  In a screening method according to one embodiment of the invention, a T7 phage, wherein a peptide comprising a sequence in which two cysteines are separated by one or more random amino acids is genetically engineered to be fused to the C-terminus of a gp10 protein. Use the library. Since this peptide fused to the C-terminus of the gp10 protein is displayed on the outer surface of the T7 phage particle (see Patent Document 1), it is referred to herein as a “displayed random peptide”. As used herein, “library” refers to a large group of phages or molecules that differ in structure. The process of selecting a phage or molecule having a specific property (for example, the ability to bind to a target protein) from a library is called “screening”.

  Random amino acid means any one of 20 amino acids selected at random, as understood by those skilled in the art. As will also be appreciated by those skilled in the art, random amino acids in the displayed random peptide are generated by randomizing the sequence of DNA encoding the amino acid and incorporating the randomized DNA sequence into the T7 phage as part of the gp10 gene. Can do. Each individual T7 phage takes in one type of gp10 gene DNA sequence and expresses one type of displayed peptide sequence, but when viewed as a whole T7 phage group, a library of random displayed peptide sequences can be obtained. . Methods of genetic manipulation to modify the sequence of the T7 phage gene and methods to generate T7 phage incorporating the modified gene are known to those skilled in the art.

The number of random amino acids inserted between two cysteines is, for example, 3 to 12, alternatively 4 to 10, preferably 5 to 9, and 7 is particularly preferable. Random amino acids may be added to the outside of the two cysteines, that is, further to the N-terminal side of the N-terminal cysteine, or further to the C-terminal side of the C-terminal cysteine, or both. The number of these outer amino acids may be, for example, 0 to 12 for each of the N-terminal and C-terminal, preferably 1 to 10, more preferably 2 to 5, and more preferably 3. The number of random amino acids on the N-terminal side and the C-terminal side may be different. As an example, it presented random ^peptide, that ^{X 1 -X 2 -X 3 -C 4} -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C 12 -X 13 -X 14 Preferably it has an amino acid sequence, wherein X ^{1 to} X ³ , X ^{5 to} X ¹¹ , X ^{13 to} X ¹⁴ are each random amino acids, and C ⁴ and C ¹² are each cysteine.

A library of T7 phage displaying a cryptand structure can be obtained by reacting a library of T7 phage displaying the displayed random peptide with a crown ether linker compound and modifying the displayed random peptide with crown ether. . This reaction is achieved by mixing the library of T7 phage displaying the displayed random peptide and the crown ether linker compound in the presence of a reducing agent such as TCEP using, for example, the 10BASE _d- T method. (Patent Document 1, Non-Patent Document 1). The cryptand library thus obtained is useful for screening cryptand molecules that bind to any target molecule.

As the crown ether linker compound, a dihalogen compound having a structure in which a haloacetyl group or a haloacetamido group is linked to two nitrogen atoms facing each other on the crown circumference of the azacrown ether can be preferably used. Non-carbon atoms other than these two nitrogens on the circumference of the crown may be oxygen or nitrogen. The crown size can be from 12-crown-4 to 30-crown-10, with 18-crown-6 being particularly preferred. More specifically, a dihalogen compound having a structure represented by any of the following formulas (VII) to (X) can be used,
In each formula, n is an integer of 1 to 4, and R ^{2 to} R ⁵ are each independently hydrogen, a hydrocarbon group having 12 or less carbon atoms, or a hydrocarbon group having 6 or less carbon atoms, The enclosed X represents halogen. Examples of the case where R ^{2 to} R ⁵ are hydrocarbon groups include substituted or unsubstituted linear, branched, or cyclic alkyl groups or aryl groups, or composite groups thereof. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, sec-butyl, cyclohexyl, phenyl and the like. The halogen can be F, Cl, Br, I or At, preferably Cl, Br or I, particularly preferably Br. After the reaction with the peptide, this halogen is replaced by a sulfur atom derived from the side chain of the cysteine of the peptide. These linker compounds can be synthesized by organic chemical techniques known to those skilled in the art using readily available material compounds.

Examples of the reducing agent that can be used in the above reaction include tris (2-carboxyethyl) phosphine hydrochloride (TCEP), dithiothreitol (DTT), β-mercaptoethanol, and the like, and TCEP is particularly preferable. For example, trialkylphosphine such as TCEP has been reported to react with haloacetic acid and its amide derivatives. For this reason, reduction and alkylation of cysteine residues are generally carried out by two independent reactions. However, in the 10BASE _d -T method, reduction and alkylation reactions can be performed in one step, that is, “one pot” without purification after reduction by TCEP (see Patent Document 1).

  The concentration of the reducing agent in the one-pot reaction can be appropriately determined by those skilled in the art and is not particularly limited, but is preferably 10 to 2000 μM, more preferably 100 to 1000 μM, still more preferably 200 to 800 μM, and most preferably 400 to 600 μM.

  Here, the reaction for preparing a library of modified T7 phages containing a cryptand structure has been described. However, in order to obtain a specific cryptand molecule or a specific crown ether modified T7 phage according to an embodiment of the present invention. Similar reactions can be used. In that case, a presentation peptide having a specific amino acid sequence may be used instead of the presentation random peptide.

  The library of modified T7 phage containing the cryptand structure obtained as described above is mixed with the target molecule and brought into contact with each other in a solution. As a result, among the phages contained in the library, the phage displaying a cryptand structure having affinity for the target molecule selectively binds to the target molecule. Specific procedures for mixing / binding and subsequent cleaning can be appropriately designed by those skilled in the art based on standard techniques, and are exemplified in Patent Document 1 and the like.

The modified T7 phage may be mixed with the target molecule as a complex state in which one metal ion is incorporated in the cryptand ring by adding a metal ion. Examples of preferred metal ions in this case include, but are not limited to, Mg ²⁺ , Fe ²⁺ , Co ²⁺ , Ni ²⁺ , Cu ²⁺ , and Zn ²⁺ .

  “Target molecule” means a molecule that is intended to be a binding target of a molecule obtained by screening. The target molecule is a molecule arbitrarily selected by the user and is typically a protein (target protein), but may also be a nucleic acid, sugar, lipid, other macromolecule, or a complex thereof. The target protein may be a full-length protein or a protein fragment. In one example, the target protein is an N-terminal domain fragment of Hsp90. The target protein may also be used for screening in a form fused to a tag of other peptides (including oligopeptides and polypeptides). Examples of such peptide tags include, but are not limited to, glutathione-S-transferase (GST), maltose binding protein (MBP), His tag, FLAG tag, and the like. The target protein may also be modified such as biotinylation. These incidental structures can facilitate the purification of the target protein and the operation of binding and elution on the column.

  Next, the target molecule and the modified T7 phage group bound to the target molecule are separated from the modified T7 phage group not bound to the target molecule by using an appropriate means according to the type of the target molecule. For example, if the target protein is in the form of a GST fusion protein, the target protein and the modified T7 phage group bound thereto are separated and recovered by affinity chromatography for glutathione, and the unbound modified T7 phage group is removed. can do. A series of these operations including mixing the phage with the target molecule and separating and recovering the bound phage from the unbound phage is referred to as “positive selection”. The phage group obtained after positive selection is a part (subgroup) of all phage groups contained in the original library.

  When the target protein is in a form fused to a tag, it is preferable to perform “negative selection” in which phages that bind to the tag portion itself are removed prior to positive selection. In the negative selection, contrary to the positive selection, the bound fraction is discarded, and the fraction not bound is collected and used in the subsequent experiments. Similarly, when a carrier such as a bead is used in the binding / washing operation, it is preferable to perform negative selection for removing phages bound to the carrier.

  The phage group separated by the positive selection can be directly infected with the host bacteria. For example, when the target protein and the phages bound thereto are separated together by utilizing the binding to the beads, the beads containing the complex of these can be used as they are for the infection reaction. Of course, as long as separation from the beads or separation from the target protein is possible, the separated phage alone may be used for infection. Any host bacterium that can be infected by T7 phage can be used, but usually E. coli (eg, BLT5403 strain) is used as the host bacterium. As an infection method, a standard method known to those skilled in the art can be used.

As will be appreciated by those skilled in the art, infection with host bacteria can propagate T7 phage displaying a peptide sequence corresponding to the cryptand structure bound to the target molecule. However, as described above, new phage particles obtained by growth do not have crown ether modification in the displayed peptide. Therefore, the propagated T7 phage was reacted with a crown ether linker compound (10BASE _d- T reaction) in the same manner as that performed on the phage library in the above, and the modified T7 phage group containing the cryptand structure was regenerated. get. The modified T7 phage group reacquired in this way is enriched with phage having the ability to bind to target molecules, compared to the original modified phage library.

  It is understood that the more positive selection is repeated, the more concentrated the phage having the ability to bind to the target molecule. Therefore, in this screening method, the modified T7 phage group reacquired above is mixed again with the target molecule, and the modified T7 phage that binds to the target molecule is separated again. That is, the second positive selection is performed. In this way, a phage containing a cryptand molecule that can bind to the target molecule can be obtained.

  Thereafter, the cycle of infection / proliferation of the separated phage, modification with the crown ether linker compound, binding to the target molecule, and separation of the bound phage is preferably repeated at least once more (3 rounds in total), at least 4 times. It is more preferable to repeat (6 rounds in total). These iterations significantly enrich phages that have the ability to bind to the target molecule and increase the rate at which molecules with higher binding power are included. The number of rounds required to isolate the desired molecule can vary depending on the type of target molecule.

  In this way, the method of concentrating phages (peptides) having binding ability by repeating positive selection and growth of phages that bind to a target is generally called “biopanning”.

  After the final positive selection, the isolated phage can be infected and propagated, and any number of clones can be isolated to confirm the binding ability. In order to confirm the binding ability, a technique known to those skilled in the art such as ELISA (Enzyme-Linked Immunosorbent Assay) may be appropriately used. Moreover, by determining the DNA sequence of the coding region terminal part of the gp10 gene in these clones, the amino acid sequence of the peptide part of the cryptand that provided the binding ability can be determined.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in detail, this invention is not limited to these Examples.

[Overview]
Of a T7 phage displaying the randomized peptide sequence X ₃ CX ₇ CX _3, where X ₃ represents 3 random amino acids, X ₇ represents 7 random amino acids and C represents cysteine The library was modified with crown ether using the 10BASE _d -T method to prepare a library of cryptand phage (cryptan dry).

  Using the modified phage described above, positive selection for the target protein GST-Hsp90 NTD (the Hsp90 N-terminal domain (NTD) fused to the GST tag) and negative selection for GST were performed, and Hsp90 NTD (PDB ID: The T7 phage having a cryptand having a binding force for 1YER) was concentrated 6 times.

Phage obtained by positive selection (0th to 6th rounds) is grown, and the suspension is subjected to a crown ether modification reaction with 10BASE _d -T, and then the binding ability to GST-Hsp90 NTD is determined by ELISA. As a result of the evaluation, the presence of a peptide binding to GST-Hsp90 NTD was observed from both unmodified peptide (mock) and cryptand (crown ether modified peptide).

A new molecule that binds from chestnut heptane drive during the rally to GST-Hsp90 NTD in order to get isolated, for 22 clones were selected at random after 6 round end of positive selection, in each 10BASE _d -T An unmodified peptide and a cryptand (crown ether modified peptide) were prepared, and the binding ability to GST-Hsp90 NTD was evaluated again by ELISA.

  As a result, one clone having a cryptand structure that strongly binds to GST-Hsp90 NTD was found out of 22 clones. The amino acid sequence of the peptide part of this cryptand structure was determined through DNA sequence analysis and compared with the sequence of a known Hsp90-binding peptide, but no commonality was found between the sequences.

[Preparation of T7 phage library]
Using the T7Select10-3b system (Merck Millipore), a T7 phage library in which the display peptide was fused to the C-terminus of gp10 was prepared according to the manual. At that time, in order to facilitate detection and analysis, a FLAG tag having the sequence of the enterokinase cleavage site (DDDDK) was inserted between gp10 and the displayed peptide. The presented peptide was designed to have two cysteine residues and a random sequence surrounding them. Briefly, first, an oligonucleotide having the following base sequence: 5′-GGTGGAGGTGGCGACTACAAGGATGACGATGACAAGGGATCA (NNK) ₃ TGC (NNK) ₇ TGT (NNK) ₃ TGAAAGCTTGGA-3 ′ (SEQ ID NO: 1) was synthesized (N is random). Nucleotide (ie, “A, C, G, or T”) and K means “G or T”). Double-stranded DNA was amplified from the oligonucleotide by PCR using appropriate two primers each containing the recognition sequences of restriction enzymes EcoRI and HindIII. The PCR product was purified and cut with EcoRI and HindIII. The DNA fragment was further purified and ligated into the T7Select10-3b vector. After packaging using this ligation product and T7 packaging extract, E. coli BLT5403 cells were infected. The quality of the T7 library was confirmed by checking the DNA sequence of randomly selected clones.

[Organic synthesis of crown ether derivatives]
According to the following scheme 1, an azacrown ether derivative as a cryptand precursor was synthesized from a commercially available compound.

0.30 g (1.1 mmol) of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane was dissolved in 2.0 mL of CH ₂ Cl ₂ and 0.60 mL in 0 ° C. ice water. Of N, N-diisopropylethylamine (DIPEA) was added and stirred for 15 minutes. Thereafter, 0.30 mL (2.3 mmol) of bromoacetyl bromide was gradually added dropwise using a syringe, and the mixture was gradually returned to room temperature and stirred for 5.5 hours. When the reaction solution was confirmed by TLC (MeOH), the product showed an Rf value of 0.7 with respect to the Rf value of 0.4 of the raw material. Since it was confirmed that the raw material disappeared, the reaction solution was concentrated under reduced pressure. The resulting crude product was reverse-phase middle pressure liquid chromatography (Yamazen ODS column 26 × 300 mm, flow rate 20 mL / min, gradient of 5-100% MeOH in pure water over 40 minutes). , Λ = 260 nm), and the target dibromo compound was obtained in a yield of 30% (0.15 g). See FIG. This dibromo compound can be used as a crown ether linker compound in the following experiments.

[Chemical modification of T7 phage with crown ether derivatives (cryptandation)]
(1) Crown ether modification to model phage Prior to the selection (molecular evolution) experiment using the library, it was examined whether the displayed peptide on the phage could be modified with crown ether. That is, the model phage (gp10-FLAG tag-GSRVS C GGRDRPG C LSV) was modified with crown ether using the 10BASE _d -T method, and then mass measurement was performed by LC-MS / MS. The specific procedure is shown below.

(2) Specific procedure for modification of crown ether with 10BASE _d -T Place a monoclonal suspension of model phage in an amount of 2.4 × 10 ¹¹ pfu in a 1.5 mL sample tube. (Use 1.2 x 10 ¹¹ pfu for each sample. Two samples of phage were prepared in advance to bisect for crown ether modification and mock modification.)
↓
↓ Add 20% PEG-6000 / 2.5M NaCl in 1/5 volume of the phage suspension.
↓ Pipetting, vortexing ↓ Centrifugation (4 ° C, 15,000 rpm, 10 minutes)
↓ Supernatant removal ↓ Add 180 μL of 1M NaCl.
↓ Sonication (5 minutes)
↓ Add 520 μL of PBS (phosphate buffered saline).
↓ Sonication (1 minute)
↓ Desktop centrifugation (room temperature, 12,000 rpm, 5 minutes)
Place 690 μL of supernatant in a new 1.5 mL sample tube.
In the following, experiments were performed using ice water at 4 ° C. without increasing the temperature.
↓ Add 40 μL of 10 mM TCEP-NaOH (pH 7).
↓ Divide into 365 μL halves. One is for crown ether modification (A), and the other is for TMRIA (tetramethylrhodamine-5-iodoacetamide, a fluorescent substance of haloacetamides) modification (B).
↓ For 365 μL of each phage suspension,
(A) Add 6.5 μL of 10 mM crown ether linker compound aqueous solution (final concentration: 174 μM).
(B) Add 65 μL of 1 mM TMRIA (final concentration: 151 μM).
↓ Stir with a rotary mixer (Nisshin Rika) (4 ℃, 3 hours)
↓ Add 22 μL of 100 mM beta-mercaptoethanol.
↓ Stir with a rotary mixer (Nisshin Rika) (4 ℃, 1 hour)
↓ Add 20% PEG-6000 / 2.5M NaCl in 1/5 volume of the phage suspension.
↓ Pipetting, vortexing ↓ Centrifugation (4 ° C, 15,000 rpm, 15 minutes)
↓ Supernatant removal ↓ Add 40 μL of standard 1 × SDS sample buffer.
↓ Sonication (5 minutes)
↓ Keep warm at 95 ℃ (5 minutes)
↓
Completed (a) crown ether modified phage library solution and (b) TMRIA modified phage library solution for electrophoresis ↓
↓ SDS-PAGE (15% acrylamide gel, 25 mA, 60 minutes) is performed using the entire amount of the completed solution.
↓ CBB (Coomassie Brilliant Blue) staining of gel (30 minutes)
↓ Gel washing (10 minutes)
↓ Shooting gel using Bio-Rad's ChemiDoc XRS PLUS

(3) Elution of the modified target product A band around 45 kDa, which is considered to be the target product, was cut out from the gel, and the gel piece was cut into a size of about 1 mm square, and then placed in a 1.5 mL sample tube.
↓ Add 100 μL of decolorizing solution and shake (37 ° C., 800 rpm, 10 minutes) × 2 times ↓ Remove decoloring solution ↓ Add 30 μL of reducing solution (60 ° C., 10 minutes)
↓ Remove the reducing solution ↓ Add 30μL of the modifying solution (room temperature, 1 hour, dark place)
↓ Remove the modifying solution ↓ Place 200μL of decolorizing solution and shake (37 ℃, 800rpm, 10 minutes)
↓ Remove decolorizing solution ↓ Add 50μL of acetonitrile (room temperature, 5 minutes)
↓ Remove acetonitrile ↓ Dry the gel piece using an evaporator (10 minutes)
↓ Add 10 μL of activated trypsin solution (room temperature, 15 minutes)
↓ Add 25 μL of digestive juice (37 ° C., 12 hours)
↓ Transfer the digestive juice mixture (A) to a new 1.5 mL tube and add 5 μL of 1% formic acid to the (A) ↓ Add 20 μL of 1% formic acid to the remaining gel piece (B) and sonicate Yes (10 minutes)
↓ Transfer only the solution fraction of (B) to (A) ↓ Confirm that the mixed solution of (A) and (B) is acidic ↓
Analysis by LC-MS / MS

The reagents used for the elution are summarized in Table 1.

(4) Results of Crown Ether Modification of Model Phage FIG. 2 shows the results of LC-MS / MS analysis of a sample obtained by applying crown ether modification to the displayed peptide on the model phage. As shown in FIG. 2, a fragment corresponding to the crown ether modified peptide was detected. From this result, it is understood that the crown ether modification performed using the 10BASE _d- T method on the displayed peptide on the model phage was successful.

[Optimization of crown ether concentration in 10BASE _d- T reaction]
(1) Specific procedure of optimization experiment When 10BASE _d -T was performed using 200 μM fluorescein iodoacetamide (FL-IA), almost all T7 phage display peptides were chemically modified by FL-IA ( > 95%). This was used to determine the optimum concentration of the crown ether linker compound in the 10BASE _d -T reaction. This technique is described in Tokunaga et al., Molecules, 19, 2481-2496 (2014). Here, an experiment similar to the procedure shown in FIG. The same model phage as described above was used. The specific procedure is shown below.

Place the amount of phage: 1.0 × 10 ¹² pfu of the model phage monoclone suspension in a 1.5 mL sample tube.
↓
↓ Add 20% PEG-6000 / 2.5M NaCl in 1/5 volume of the phage suspension.
↓ Pipetting, vortexing ↓ Centrifugation (4 ° C, 15,000 rpm, 10 minutes)
↓ Supernatant removal ↓ Add 180 μL of 1M NaCl.
↓ Ultrasonic (5 minutes)
↓ Add 520 μL of PBS.
↓ Sonication (1 minute)
↓ Desktop centrifugation (room temperature, 12,000 rpm, 5 minutes)
Place 690 μL of supernatant in a new 1.5 mL sample tube (hereinafter referred to as phage solution).
In the following, experiments were performed using ice water at 4 ° C. without increasing the temperature.
Prepare 8 samples of a mixture of the following composition in a 1.5 mL tube.

Phage solution: 80 μL
10 mM TCEP-NaOH 5 μL
10 mM crown ether linker compound aqueous solution X μL
PBS… the rest
-------------------------------------------------- ----------------------
Total: 100 μL

In order to vary the crown ether concentration between 0 μM and 1 mM, X = 0, 0, 1, 2, 4, 6, 8, and 10. (Two samples with X = 0 μL were prepared since a negative control not modified with FL-IA was required later.)
↓ Stir with a rotary mixer (Nisshin Rika) (4 ℃, 3 hours)
↓ Add 10 μL of 2 mM FL-IA aqueous solution / DMSO = 1: 1 solution.
↓ Stir with a rotary mixer (Nisshin Rika) (4 ℃, 3 hours)
↓ Add 5 μL of 100 mM beta mercaptoethanol.
↓ Stir with a rotary mixer (Nisshin Rika) (4 ℃, 30 minutes)
↓ Add 20% PEG-6000 / 2.5M NaCl in 1/5 volume of the phage suspension.
↓ Pipetting, vortexing ↓ Centrifugation (4 ° C, 15,000 rpm, 15 minutes)
↓ Supernatant removal ↓ Add 40 μL of 1 × SDS sample buffer ↓ Sonication (5 minutes)
↓ Keep warm at 95 ℃ (5 minutes)
↓ Centrifugation (room temperature, 12,000 rpm, 5 minutes)
↓ SDS-PAGE using 15 μL of the completed solution (10% acrylamide gel, 25 mA, 60 minutes)
↓
Photographing gels using Bio-Rad ChemiDoc XRS PLUS and Hitachi Solutions FMBIO (registered trademark) III

(2) Result of optimization experiment As described above, the result of detecting fluorescence derived from FL-IA by electrophoresing a gp10 fusion peptide first modified with different amounts of crown ether and then with FL-IA ( FIG. 3A shows a gel photographed with ChemiDoc XRS PLUS. A graph of the fluorescence intensity of FL-IA is shown in FIG. 3B (quantified after scanning the gel with FMBIO III).

As shown in FIGS. 3A and 3B, as the crown ether concentration is increased, the fluorescence intensity decreases (that is, crown ether modification increases in a concentration-dependent manner, and there is less room for modification of FL-IA added later). ) Furthermore, it can be seen that when the crown ether concentration is increased to 400 μM, the fluorescence intensity is almost lost (that is, almost all of the presenting peptides have been modified with the crown ether). From the above results, the optimum concentration of the crown ether linker compound in 10BASE _d -T was determined to be 500 μM (with some margin). In the screening described below, the concentration of the crown ether linker compound during the 10BASE _d -T reaction was 500 μM.

[Screening of Hsp90-binding cryptands using a library of cryptanded T7 phage]
A cryptand library was prepared by performing crown ether modification by the 10BASE _d- T method on the T7 phage library described above. Using this cryptand library as an initial library, a cryptand structure that specifically binds to the target protein Hsp90 by performing biopanning by repeating growth and re-modification of phage groups that have undergone positive selection against GST-Hsp90 NTD Were sequentially enriched and screened (note that the phage binding to these parts was eliminated in advance by performing a negative selection on GST and glutathione beads). In this example, a total of 6 rounds of positive selection were performed. Finally, the sequence of the displayed peptide on the phage was analyzed by determining the DNA sequence of the phage clone whose binding was confirmed by ELISA assay. An outline of these series of procedures is shown in FIG.

[Results of binding ability evaluation by ELISA]
For samples that were infected and propagated after 0-6 rounds (R0-R6) of positive selection, perform crown ether modification using 10BASE _d- T again (in this case, an unmodified sample is also prepared as a negative control) The binding ability to the target protein GST-Hsp90 NTD or the control protein BSA (bovine serum albumin) was evaluated by ELISA. FIG. 5 shows the measurement results of the color intensity representing the binding amount in this assay.

  According to FIG. 5, it can be seen from the fifth round (R5) that the phages that bind to GST-Hsp90 NTD are rapidly concentrated in the unmodified phage group. The crown ether-modified phage group is significantly enriched from round 6 (R6), but the polyclonal binding is weaker than the unmodified phage group (ie, phage rather than individual phage). The binding amount as a whole group is considered to be lower).

However, it can be expected that the cryptand molecules having strong binding power as individual monoclones are contained in the phage group modified with crown ether. Therefore, 22 monoclones were randomly selected from the phage plaques obtained in the titer check immediately after the 6th round, numbered, re-modified with 10BASE _d- T, and each clone again. The binding ability was evaluated by ELISA. The color intensity measured by this ELISA represents the strength of the binding force as a monoclone. The measurement results are shown in FIG.

  From FIG. 6, it was confirmed that the crown ether modified No. 18 monoclone had a strong binding force to GST-Hsp90 NTD.

[Analysis of peptide sequences via DNA sequencing]
Using the 18th monoclone confirmed to bind to GST-Hsp90 NTD by modification with crown ether, DNA amplification of the gp10 gene was performed by PCR, and the PCR product was purified. Then, the DNA sequence of the presented peptide was determined. As a result, it was revealed that the displayed peptide of this clone had the following amino acid sequence.
Presented peptide sequence of clone 18:
QWV C LNPWLSI C RA
The underlined cysteine is an amino acid that was not randomized in the initial library, and the side chain of this cysteine is linked to the crown ether.

On the other hand, according to research to date, peptides having the following sequences are known as peptides that bind to Hsp90 NTD (see Non-Patent Document 1). Note that □ indicates any amino acid. As described above, the cysteine indicated by underlining is a non-randomized amino acid, and actually these two cysteines form a disulfide bond.
Prior art Hsp90 binding peptide sequences:
R □ T C Y □□□□□□ C □□ W

And the arrangement | sequence of the peptide part of the Hsp90 NTD binding cyclic molecule (non-cryptand) described in patent documents 1 and non-patent documents 1 was as follows.
Sequence of the peptide portion of the prior art Hsp90 binding cyclic molecule:
RSW C RKSRKNSGGGLVW C F

  Comparing the above three amino acid sequences, no commonality other than cysteine is found. Therefore, unlike the case of the known Hsp90 NTD-binding peptide, in the present invention, a unique hard cryptand-like structure formed by combining the crown ether and the peptide works favorably for binding to Hsp90. This is thought to have led to the identification of a completely new sequence.

[Chemical synthesis of Hsp90-binding cryptand and measurement of various physical properties]
A peptide having the same sequence (H ₂ N-QWVCLNPWLSICRA-OH) as the 18th clone was commissioned to GenScript (NJ, USA) and synthesized and analyzed. The purity of the synthetic peptide was estimated to be over 90%. In order to cyclize the azacrown ether derivative (dibromide compound) shown in the above scheme 1 together with this peptide, the peptide (12 mg, 7.1 μmol) was dissolved in 70 mL of phosphate buffer (10 mM Na phosphate, pH 7.4). Then, an aqueous solution having a final concentration of 0.10 mM was prepared, and then dibromide compound (17 mg, 0.48 mM) dissolved in a small amount of water and neutralized TCEP (0.50 mM) were gradually added over 5 minutes. This mixture was incubated for 3 hours while shaking in the dark at room temperature to carry out the reaction. To inactivate the remaining dibromide, a 2-mercaptoethanol stock solution (5.0 μL, 1.0 mM) was further added to the reaction mixture and incubated for an additional 30 minutes with shaking in the dark at room temperature. The mixture was then lyophilized. The lyophilizate was dissolved in an aqueous solution of 1% formic acid / 25% DMSO and the product of the above reaction was performed using a reverse phase HPLC (Shimadzu Corporation) equipped with an XTerra Prep MS C18 column (10 × 50 mm, Waters). A cryptand molecule was purified. Cryptand molecules were separated at a flow rate of 4 mL / min using a 0-100% linear acetonitrile gradient containing 0.1% formic acid over 50 minutes. The resulting cryptand molecule (5.0 mg, 35% yield) was lyophilized and analyzed by LC-MS / MS and NMR.

FIG. 7 shows the LC-MS / MS spectrum of the cryptand molecule analyzed using a 0-100% linear methanol gradient over 50 minutes with 0.1% formic acid. (A) shows total ion chromatography, (B) shows an MS spectrum around 29.6 minutes, and (C) shows an MS / MS spectrum of a fragment having an m / z of 1016.8. Prior to circularization, most of b and y MS / MS fragment of linear peptide _{(H 2 N-QWVCLNPWLSICRA-OH} ) was identifiable (data not shown), after cyclization, limited Only a few MS / MS fragments are found.

[CD spectrum of Hsp90 NTD-binding cryptand]
CD (circular dichroism) spectra were measured for both the linear linker peptide (that is, the peptide before crown ether modification) and the cryptand. Briefly, each compound was dissolved in a phosphate buffer (20 mM phosphate, pH 7.4) to a final concentration of 20 μM, and a circular dichroism dispersometer (J-720W, manufactured by JASCO) at 25 ° C. and The spectrum was measured using a 1 cm optical path length quartz cell.

  In both the linear peptide and the cryptand, characteristic peaks similar to each other were observed (FIG. 8). This result suggests that the secondary structure of the peptide is retained as part of a rigid cyclic structure rather than being destroyed by the crown ether bond. This is in contrast to the prior art non-cryptand-type crown analogs that have shown high structural flexibility (Non-Patent Document 1), and the cryptand molecule of the present invention exhibits a strong binding force. This is thought to be the cause.

[Isothermal titration calorimetry (ITC)]
The ITC experiment was performed using MicroCal iTC ₂₀₀ (manufactured by GE Healthcare). GST-tagged Hsp90 NTD was concentrated using an ultrafiltration column (vivaspin column 500, molecular weight cut-off 10 kDa, manufactured by GE Healthcare), and the buffer was phosphate buffer (20 mM phosphate-KOH, pH 7.2, 50 mM). (NaCl, 1 mM TCEP). Protein concentration was determined by Bradford assay. For titration experiments, GST-Hsp90 NTD or GST and cryptand were diluted to 5 μM and 50 μM, respectively. Titration was performed at 25 ° C. The injection parameters were as follows: volume 2 μL, duration 4 seconds, interval 60 seconds, and filter time 5 seconds. The reference power was set to 5 μcal / s. Data was analyzed using Origin Software 7.0 (MicroCal). Curve fitting was performed using a 1: 1 interaction model.

The results of the ITC experiment (FIG. 9) clearly show that this cryptand molecule binds specifically to Hsp90 NTD and not to GST. The data obtained for binding to GST is disordered indicating the lack of specific interactions. Dissociation constant, K _D, obtained for binding to Hsp90 NTD is was 62 nM, which is far in exceeds the non-cryptand cyclic molecules of the prior art (Non-Patent Document 1), a strong bonding force as comparable to an antibody Is a numerical value representing In addition, the enthalpy change ΔH is larger (−16.9 kcal / mol) than the value (−13.7 kcal / mol) obtained with the above-described prior art cyclic molecule. This suggests that this cryptand molecule according to embodiments of the present invention is relatively susceptible to hydrogen bonding with the target protein. Since -TΔS = 7.0 and the measurement temperature is 25 ° C., the entropy change ΔS is calculated as −7000 / (273 + 25) = − 23.5 cal / mol / deg. This is a value that is similar to that of Non-Patent Document 1 as compared with -19.6 cal / mol / deg, or rather is slightly disadvantageous in terms of entropy. Compared to the crown-like molecule of Non-Patent Document 1, in this cryptand, which is a bicyclic structure that is smaller and more rigid, the entropy loss associated with the bond is slightly increased (that is, the entropy is very slight). The disadvantage is that while entropy loss (disadvantage) occurs due to an increase in the number of hydrogen bonds associated with the target molecule, the entropy loss is reduced (advantage) ) Offset this, and as a result, it is presumed that ΔS becomes almost the same value.

[Nuclear magnetic resonance (NMR) measurement]
Use 500MHz spectrometer (JNM-ECA500, Jeol Resonance Ltd.), at 30 ° ^C., was subjected to NMR experiments involving 1 ^H- 1 H COZY and STD (Saturation Transfer Difference) measurements. The assignment of the normal one-dimensional proton NMR ( ¹ H NMR) spectrum of the cryptand is the various two-dimensional NMR spectra (specifically, ¹ H- ¹ H COSY, TOCSY (mixing time 150 milliseconds), NOESY (mixing time 0). .5 seconds)) was measured and comprehensively analyzed. All NMR spectra were measured by dissolving cryptand (0.32 mM) in 10 mM phosphate buffer (pH 7.4) prepared with 90% D ₂ O and 10% DMSO-d ₆ as the solvent. Saturation transfer difference NMR spectrum (STD-NMR) was measured by irradiating only GST-Hsp90 NTD protein with a Gaussian pulse of 60 milliseconds, with a saturation time of 4 seconds. The on-resonance and off-resonance pulse irradiation positions for the GST-Hsp90 NTD protein were 0.5 and -10 ppm, respectively, and the STD-NMR spectrum was obtained by taking the difference between them. The total number of integrations during the STD-NMR spectrum measurement is 7432, and the number of domain data points is 4096.

  The results of STD-NMR (FIG. 10) show that among the peptide parts of the cryptand molecule according to the embodiment of the present invention, hydrophobic amino acids excluding alanine (A), that is, tryptophan (W), leucine (L), isoleucine (I) And valine (V) has been shown to play a particularly important role in binding to Hsp90.

Claims (21)

A molecule having a structure represented by the following formula (I):
Where
X ¹ to X ¹⁴ (including ^{C 4} and ^{C 12)} represents amino acids joined by peptide bonds, ^{C 4} and ^{C 12} each represents a cysteine, ^{S *} is derived from cysteine ^{C 4} and ^{C 12} Represents a sulfur atom,
X ¹ is any amino acid,
X ² is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ³ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁵ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁶ is any amino acid,
X ⁷ is any amino acid,
X ⁸ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁹ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ¹⁰ is any amino acid,
X ¹¹ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ¹³ is any amino acid,
X ¹⁴ is any amino acid or absent,
R ¹ is a group having a structure represented by any of the following formulas (II) to (V):
Where
n is an integer of 1 to 4,
R ^{2 to} R ⁵ are each independently hydrogen or a methyl group.
molecule. X ² is tryptophan,
X ³ is valine,
X ⁵ is leucine,
X ⁸ is tryptophan,
X ⁹ is leucine,
X ¹¹ is isoleucine,
The molecule of claim 1. The molecule according to claim 1 or 2, wherein X ⁷ is proline. X ¹ , X ⁶ , X ¹⁰ , and X ¹³ are each independently a hydrophilic amino acid selected from the group consisting of glutamine, asparagine, serine, threonine, arginine, lysine, histidine, tyrosine, aspartic acid, and glutamic acid. The molecule according to any one of claims 1 to 3, wherein X ¹ is glutamine or asparagine,
X ⁶ is glutamine or asparagine,
X ¹⁰ is serine or threonine;
X ¹³ is arginine or lysine,
The molecule according to any one of claims 1 to 4. The peptide chain portion represented by X ^{1 to} X ¹⁴ (including C ⁴ and C ¹² ) is
QWVCLNPWLSICRA
Having an amino acid sequence of
The molecule according to any one of claims 1 to 5. The molecule according to claim ^1, wherein in R ¹ , n = 2. R ¹ is a group having a structure represented by the following formula (II):
Where n = 2.
The molecule according to any one of claims 1 to 7. A molecule having a structure represented by the following formula (VI).
The following sequence ^{X 1 -X 2 -X 3 -C 4} -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C 12 -X 13 -X 14
A composition for producing an hsp90 binding agent comprising a peptide having:
X ^{1 to} X ¹⁴ (including C ⁴ and C ¹² ) represent amino acids linked by peptide bonds, C ⁴ and C ¹² each represent cysteine,
X ¹ is any amino acid,
X ² is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ³ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁵ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁶ is any amino acid,
X ⁷ is any amino acid,
X ⁸ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ⁹ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ¹⁰ is any amino acid,
X ¹¹ is a hydrophobic amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine, and methionine;
X ¹³ is any amino acid,
X ¹⁴ is any amino acid or absent,
The peptide is linked to the two nitrogen atoms of the azacrown ether via the C ⁴ and C ¹² cysteine side chains to yield an hsp90 binding agent,
Composition. X ² is tryptophan,
X ³ is valine,
X ⁵ is leucine,
X ⁸ is tryptophan,
X ⁹ is leucine,
X ¹¹ is isoleucine,
The composition according to claim 10. The composition according to claim 10 or 11, wherein X ⁷ is proline. X ¹ , X ⁶ , X ¹⁰ , and X ¹³ are each independently a hydrophilic amino acid selected from the group consisting of glutamine, asparagine, serine, threonine, arginine, lysine, histidine, tyrosine, aspartic acid, and glutamic acid. The composition according to any one of claims 10 to 12, which is X ¹ is glutamine or asparagine,
X ⁶ is glutamine or asparagine,
X ¹⁰ is serine or threonine;
X ¹³ is arginine or lysine,
The composition according to any one of claims 10 to 13. The peptide chain portion represented by X ^{1 to} X ¹⁴ (including C ⁴ and C ¹² ) is
QWVCLNPWLSICRA
Having an amino acid sequence of
The composition according to any one of claims 10 to 14.   A T7 phage, wherein the peptide portion of the molecule according to any one of claims 1 to 9 is fused to the C-terminus of the gp10 protein. A method of screening for molecules that can bind to a target molecule,
(A) genetically engineered so that a peptide containing two cysteines separated by 4-10 random amino acids is fused to the C-terminus of the gp10 protein; and
Live of modified T7 phage comprising a structure in which —CO—CH ₂ — groups linked to two opposite nitrogen atoms on the periphery of azacrown ether are respectively bonded to sulfur atoms of side chains of the two cysteines Rally,
Mixing with the target molecule;
(B) separating the modified T7 phage bound to the target molecule;
(C) infecting a host bacterial cell with the isolated modified T7 phage, and propagating the corresponding T7 phage;
(D) reacting the T7 phage grown in the step (c) with a dihalogen compound having a structure in which a haloacetyl group or a haloacetamide group is linked to the two nitrogen atoms of the azacrown ether, Obtaining a new phage;
(E) mixing the modified T7 phage newly obtained in the step (d) with the target molecule, and (f) separating the modified T7 phage bound to the target molecule,
Including a method. After step (f)
The method according to claim 17, further comprising (g) repeating the steps (c) to (f).   The method according to claim 18, wherein in the step (g), the steps (c) to (f) are repeated at least four times. Prior to step (a), the method further comprises an initial step of creating a library of the modified T7 phage,
A library of T7 phage genetically engineered such that a peptide containing two cysteines separated by 4-10 random amino acids is fused to the C-terminus of the gp10 protein,
The method according to any one of claims 17 to 19, comprising reacting with a dihalogen compound having a structure in which a haloacetyl group or a haloacetamide group is linked to the two nitrogen atoms of the azacrown ether. A gene engineered such that a peptide containing two cysteines separated by 4-10 random amino acids is fused to the C-terminus of the gp10 protein; and
Live of modified T7 phage comprising a structure in which —CO—CH ₂ — groups linked to two opposite nitrogen atoms on the circumference of azacrown ether are respectively bonded to sulfur atoms of the side chains of the two cysteines Rally.