WO2012074130A1 - ペプチドライブラリーの製造方法、ペプチドライブラリー、及びスクリーニング方法 - Google Patents
ペプチドライブラリーの製造方法、ペプチドライブラリー、及びスクリーニング方法 Download PDFInfo
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- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/67—General methods for enhancing the expression
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
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- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
Definitions
- the present invention relates to a method for constructing a library composed of aggregates of peptidic molecules containing a special amino acid as part of the sequence, the constructed library, and a method for screening active peptides from the library.
- Low molecular weight compounds that have a similar structure to the enzyme substrate and bind to the active pocket of the enzyme are widely used as inhibitors of the enzyme, but there are multiple enzymes with similar active pockets in the body, It is known that such a low molecular weight compound alone is not likely to be a specific inhibitor.
- peptide molecules typified by antibodies are attracting attention as next-generation pharmaceuticals because they can recognize the molecular surface in a wide range and can specifically bind to a target.
- the various in vitro display methods such as are excellent in that a highly diverse library can be constructed and screened in a short time in a single tube.
- the In vitro display method displays the phenotype as a genotype by linking the phenotype (phynotype) and the genotype encoding the sequence (genotype) by non-covalent or covalent bonds, and is reconstructed in a test tube. This refers to a system that enables the active species to be concentrated and amplified (selected) using a replication system.
- a phage display using E. coli as a replication medium can select a 10 7 diversity library, but a In vitro display can display a 10 12 diversity library. Search is possible.
- In-vitro displays include ribosome displays, mRNA displays, RaPID displays (PCT / JP2010 / 68549, an unpublished international patent application), and the like.
- mRNA display will be described below as an example.
- the mRNA display method is a technique for associating an amino acid sequence of a polypeptide with a nucleic acid sequence by binding the polypeptide and mRNA as its template.
- puromycin the terminal analog of acylated tRNA
- a peptide molecule as a translation product is linked to mRNA via puromycin (Patent Documents 1 to 3, Non-Patent Documents 1 and 2).
- in vitro display can screen peptide libraries with as many as 10 12 divergences, but in order to construct peptide libraries using the functions of living organisms, conventional protein properties Only a peptide library composed only of amino acids has been constructed. If we can overcome the low level of this component, incorporate a small molecule inhibitor with insufficient inhibitory ability and specificity into the amino acid structure, construct a peptide library containing the special amino acid, and conduct screening It can be expected that it will be possible to obtain inhibitors exhibiting high inhibitory potency and selectivity that cannot be achieved with molecular compounds and peptides alone.
- the first is the construction of an N-methylphenylalanine-containing peptide library by R. Roberts et al. (Non-patent Document 3) and screening using mRNA display.
- N-methylamino acid was designed to appear with a certain probability in a random peptide sequence.
- all the peptides obtained as a result of screening targeting G protein were all normal.
- a peptide composed only of 20 kinds of amino acids and containing N-methylphenylalanine has not been obtained.
- the second report was the construction of a peptide library incorporating sulfotyrosine and screening using phages by P.G.
- Non-Patent Document 5 Construction of a peptide library containing a special amino acid having a boron functional group that binds to a sugar and screening using a phage It is carried out. First, by constructing a phage displaying an scFv containing a random region designed so that the above-mentioned special amino acid appears with a certain probability, this special amino acid is screened against a carrier on which a sugar is immobilized. Has succeeded in obtaining an array containing 1 or 2. However, no knowledge has been obtained that specific sugars or sugar chains have specificity, and the boron functional group has a characteristic of covalently bonding to the hydroxyl group of the sugar in the first place, so it has nonspecific binding. The possibility cannot be denied. In other words, no technology has been developed for obtaining peptide sequences that bind to a sugar chain or glycoprotein having some biological significance to the obtained scFv.
- any of the above technologies is limited to the construction of a library containing only one type of special amino acid, and a peptide containing a special amino acid having the desired physiological function has not been obtained. In terms of reliability, this is an immature technology.
- Patent No. 3683282 Publication WO98 / 16636
- Patent No. 3683902 public information (International Publication WO98 / 31700)
- Patent No. 3692542 Publication WO98 / 16636
- An object of the present invention is to provide a method for constructing a peptide library composed of peptides in which amino acids having a portion capable of binding to a target substance are arranged at desired positions in a random sequence.
- the present inventors prepared an mRNA library containing a base sequence in which a modified codon specifying an amino acid capable of binding to a target substance is arranged in a base sequence encoding a random amino acid sequence, while corresponding to the modified codon.
- the present invention [1] A method for producing a peptide library consisting of peptides in which a special amino acid having a moiety capable of binding to a target substance is arranged at a designated position in a random sequence by translation, (I) a step of preparing an mRNA library comprising a base sequence in which a modified codon that designates a special amino acid having a portion capable of binding to the desired target substance is arranged in an mRNA sequence encoding a random amino acid sequence
- (Iii) translating the mRNA in a cell-free translation system containing the aminoacyl-tRNA to obtain a library comprising a collection of peptides in which a predetermined special amino acid is arranged in a random sequence
- the aminoacyl tRNA is prepared by transferring a special amino acid having a mo
- the modified codon specifying a special amino acid having a moiety capable of binding to the desired target substance is an AUG codon
- the mRNA random sequence is NNC and NNU (N is any one of A, U, G and C)
- N is any one of A, U, G and C
- the method according to [1] or [2] above comprising repeating a triplet of any one of [4]
- the method according to [3] above, wherein the mRNA random sequence further contains NNK (K represents U or G);
- the step of cyclizing each of the peptides includes: In the step (i), in the random sequence of mRNA, second and third modifications encoding two amino acids each having functional groups 1 and 2 of any of the following groups (A) to (C): A codon is arranged (however, when the amino acid having the functional group 2 is a proteinous amino acid, a codon that designates the proteinaceous amino acid may be arranged instead of the third modified codon).
- step (ii) an aminoacyl tRNA in which the amino acid having the functional group 1 is linked to the tRNA designated as the second modified codon, and the amino acid having the functional group 2 in the tRNA designated as the third modified codon And an aminoacyl-tRNA ligated with the above, using these also to perform the step (iii),
- the method according to the above [5] comprising cyclization by a reaction between the functional groups (Wherein X 1 is Cl, Br or I, and Ar is an optionally substituted aromatic ring); [7] The method according to any one of [1] to [6] above, wherein the special amino acid having a moiety capable of binding to the desired target substance is a low molecular compound-containing special amino acid; [8]
- the target substance is an enzyme, and the portion capable of binding to the target substance is a low molecular weight group that is predicted to bind to the active site of the enzyme, any one of [1] to [6] above The method according to paragraph; [
- a library composed of peptides in which amino acids into which a moiety capable of binding to a target is introduced at a desired position of a random amino acid sequence.
- By screening using such a library it is possible to search for peptides having functions that cannot be expressed alone, such as increased affinity or specificity for the target as compared with the case where a portion capable of binding is used alone.
- the diversity can be extremely high (for example, 10 to the 12th power or more), and when combined with an in vitro display method, it has an affinity for a target. It is also possible to efficiently concentrate and identify highly peptide sequences.
- a library comprising a set of peptides into which special amino acids incorporating a low molecular weight compound that can function as an enzyme inhibitor are introduced.
- screening this library it is possible to obtain inhibitors that exhibit high inhibitory ability and selectivity, not just aptamers (binding active species).
- a sequence of three bases (triplet) of mRNA designates one amino acid as one codon, and the corresponding peptide is synthesized.
- the association between the codon and the amino acid is performed in the following two stages.
- the amino acid corresponding to aminoacyl-tRNA synthetase (ARS) is linked to the end of tRNA.
- ARS aminoacyl-tRNA synthetase
- amino acids on tRNA are polymerized according to the information of mRNA, and a peptide is synthesized.
- the correspondence between such codons and anticodons is almost universally determined, and any one of 20 amino acids is assigned to each of 64 types of codons.
- the universal genetic code table is shown below.
- the genetic code can be reprogrammed by using a reconstituted translation system and an artificial aminoacylated RNA-catalyzed flexizyme.
- a reconstituted translation system is a translation system that isolates, purifies, and mixes factors involved in translation synthesis of proteins and peptides, such as ribosomes, translation factors, tRNAs, amino acids, and energy sources such as ATP and GTP. It is.
- the technique described in the following literature is known as a system using the ribosome of E. coli: HF Kung, B. Redfield, BV Treadwell, B. Eskin, C. Spears and H. Weissbach (1977) “DNA-directed In vitro synthesis of beta-galactosidase.
- flexizyme is an artificial RNA catalyst (RNA catalyst having acyl-tRNA synthetase-like activity) that can link (acylate) any amino acid or hydroxy acid to any tRNA.
- RNA catalyst having acyl-tRNA synthetase-like activity
- RNA catalyst having acyl-tRNA synthetase-like activity
- RNA catalyst having acyl-tRNA synthetase-like activity
- the flexizyme is also known by the name of the original flexizyme (Fx) and dinitrobenzyl flexizyme (dFx), enhanced flexizyme (eFx), amino flexizyme (aFx) and the like modified from the original flexizyme (Fx).
- a translation system is used in which constituent elements of the translation system are freely removed according to the purpose and only necessary components are reconfigured. For example, when a translation system from which a specific amino acid is removed is reconfigured, the codon corresponding to the amino acid becomes an empty codon. Subsequently, using flexizyme, chemical aminoacylation, or aminoacylation using a mutant protein enzyme, a special amino acid is linked to a tRNA having an anticodon complementary to the vacant codon, and this is added to perform translation. . As a result, a special amino acid is encoded by the codon, and the peptide into which the special amino acid is introduced instead of the removed amino acid is translated.
- a special peptide having a chloroacetyl group at the N-terminus is synthesized by the above-described genetic code reprogramming technique.
- the mercapto group spontaneously undergoes nucleophilic attack on the chloroacetyl group after translation, and the peptide is cyclized by a thioether bond. That is, by introducing a set of functional groups capable of forming a bond between a chloroacetyl group and a mercapto group into an amino acid sequence, a function of cyclization is given to the peptide.
- the pair of functional groups capable of such bond formation is not limited to a chloroacetyl group and a mercapto group. Details will be described later.
- the peptide library constructed according to the present invention is characterized by comprising a collection of peptides having a special amino acid having a moiety capable of binding to a target substance (hereinafter referred to as “the present special amino acid”) at a desired position.
- the peptide library containing this special amino acid is constructed by artificially assigning this special amino acid to an existing codon by in vitro translation synthesis using reprogramming of the genetic code. Specifically, an mRNA library having a codon encoding this special amino acid is prepared and translated under the modified genetic code table, so that the modified codon (modified codon) is located at the designated position. A library comprising peptides into which this special amino acid has been introduced can be obtained.
- the length of the peptides constituting the peptide library is not particularly limited, and can be 2 to 25 amino acids, for example.
- a codon refers to both a modified codon and a universal codon used in natural translation.
- a modified codon refers to a codon that has been uncorrelated with a protein amino acid by genetic code reprogramming and associated with a special amino acid. Special amino acids can only be encoded with modified codons.
- the special amino acid refers to all amino acids having different structures from the 20 types of proteinic amino acids used in natural translation, and even those that are artificially synthesized exist in nature. There may be. In other words, non-protein amino acids, artificial amino acids, D-form amino acids, N-methyl amino acids, N-acyl amino acids, ⁇ -amino acids, and amino acid skeletons in which part of the side chain structure of protein amino acids is chemically modified or modified And all derivatives having a structure in which the amino group or carboxyl group is substituted.
- the special amino acid is also used as “present special amino acid” and can also be used in the cyclization method described later.
- This special amino acid has a moiety that can bind to the target substance.
- the portion capable of binding to the target substance can be predicted by various methods based on the structure of the target substance and empirical rules.
- the method for predicting the portion that can bind to the target substance is not particularly limited. For example, prediction is performed by searching a database of interaction between a receptor and a ligand, or using various software that predicts the structure of a ligand by using the receptor structure. It can also be predicted based on molecules that bind to similar target substances.
- the portion that binds to the target substance may be a part that increases or decreases the activity of the target substance, not just binding.
- the part capable of binding to the target substance may be a part originally possessed by the special amino acid, or may be a part artificially introduced into the special amino acid.
- the special amino acid can be selected from known special amino acids having a moiety capable of binding to the target substance, and in the latter case, the special amino acid binds to the target substance. What introduced the part which can be used can be used. Those skilled in the art can appropriately select and introduce these by a known method or a method analogous thereto.
- this special amino acid is a special amino acid incorporating a predetermined low molecular weight compound.
- such special amino acids are referred to as “low molecular weight compound-containing special amino acids”.
- a specific example of the low molecular weight compound-containing special amino acid is a special amino acid having a structure of a low molecular weight compound that binds to the enzyme active site of the target enzyme.
- the low molecular weight compound-containing special amino acid is arranged at a position designated by the modified codon in the random amino acid sequence.
- the peripheral sequence of the low molecular weight compound-containing special amino acid is randomized.
- small molecule is used in its broadest sense, and its structure and molecular weight are not particularly limited as long as it is originally contained in a special amino acid or can be bound to a special amino acid.
- a molecule of about 1000 Da is mentioned.
- the low molecular weight compound-containing special amino acid can be appropriately prepared by a method known to those skilled in the art or a method analogous thereto. For example, a structure of a low molecular compound that can bind to a target substance is predicted, and a special amino acid having all or part of the low molecular compound may be selected and used. Moreover, you may couple
- the library of the present invention is a peptide library in which a special amino acid containing a low molecular weight compound capable of binding to the enzyme active site is introduced in order to obtain a peptide having an inhibitory activity rather than a simple aptamer It may be a rally.
- the property that a low molecular weight compound, a low molecular weight compound-containing special amino acid, or a peptide containing such a special amino acid binds to the enzyme active site may be expressed as enzyme active site directivity.
- the structure that binds to the enzyme active site of the target enzyme is theoretically designed based on the structure of the original substrate of the enzyme, or a small molecule inhibitor that binds to the active pocket and inhibits enzyme activity is known. For example, you may decide based on it.
- low molecular weight compounds are molecules for which the specificity of physiological activity to a target substance cannot be sufficiently obtained by themselves, or molecules whose activity can still be expected to improve.
- NAD + and epsilon-N-acetyl lysine residue of the substrate protein bound to deacetylated reaction proceeds, NAD + or Analogs of ⁇ -N-acetyllysine residues can be used as inhibitors.
- the most typical inhibitors are those containing nicotinamide, which is part of the structure of NAD + , and ⁇ -N-trifluoroacetyl lysine and ⁇ -N-thioacetyl lysine.
- all inhibitors have limitations in terms of specificity between isoforms and inhibitory ability.
- the peptide library of the present invention is (I) preparing an mRNA library comprising a base sequence in which a modified codon that designates an amino acid having a portion capable of binding to the desired target substance in an mRNA sequence encoding a random amino acid sequence is disposed; (Ii) preparing an aminoacyl-tRNA in which the special amino acid is linked to a tRNA designated as a modified codon; (Iii) translating the mRNA in a cell-free translation system containing a tRNA linked to the special amino acid to obtain a library comprising a collection of peptides in which a predetermined special amino acid is arranged in a random sequence; Manufactured by the method of including.
- the order of step (i) and step (ii) is
- a translation system is a place for peptide translation synthesis and is generally a concept including both a method and a kit (product).
- the cell-free translation system used for the preparation of the special peptide library may be a known reconstitution-type translation system, or further subdivided to construct a system with fewer impurities. May be used.
- the specific components of the translation system as a kit (product) that can be used in the present invention will be described while comparing with a conventional system.
- components of the translation system include ribosome, translation initiation factor (IF) group, elongation factor (EF) group, termination factor (RF) group, ribosome regeneration factor (RRF), and at least necessary for target peptide synthesis.
- IF translation initiation factor
- EF elongation factor
- RF termination factor
- RRF ribosome regeneration factor
- Natural amino acids tRNAs, a set of specific ARS protein enzymes, and energy sources for translation reactions.
- ribosome a ribosome isolated and purified from E. coli is preferably used.
- translation initiation factors eg IF1, IF2, IF3
- translation elongation factors eg EF-Tu, EF-Ts, EF-G
- translation termination factors eg RF1, RF2, RF3, RRF
- Enzymes for energy source regeneration eg creatine kinase, myokinase, pyrophosphatase, nucleotide-diphosphatase kinase
- addition of an enzyme for regeneration of a translation termination factor / energy source is optional.
- T7 RNA polymerase may be added to perform transcription from the template DNA, but RNA polymerase need not be added when pre-transcribed mRNA is added to the translation system.
- NTPs as energy sources for translation reactions
- CreatineCphosphate CreatineCphosphate
- ribosome activation RNA stabilization
- factors necessary for protein stabilization etc.
- N-formylmethionine is defined in the start codon AUG by the start tRNA, so 10-formyl-5,6,7,8-tetrahydroforlic acid (Baggott et al., 1995)
- a formyl donor is optional when a translation reaction is initiated with a special amino acid.
- methionyl-tRNA-formyltransferase (MTF) is not essential.
- corresponding natural tRNA and ARS can be used for natural proteinaceous amino acids as in the conventional system.
- An example of a natural tRNA is a mixture obtained by collecting E. coli, crushing, and purifying a tRNA fraction therefrom, and a commercially available product is also available.
- Certain A, U, C, and G in natural tRNA are chemically modified by enzymes.
- an artificial tRNA that is a tRNA transcript as an orthologous tRNA instead of a natural tRNA.
- Artificial tRNA can be prepared by in vitro transcription using DNA as a template and an appropriate RNA polymerase. There is no chemical modification in such artificial tRNA.
- an ortho-tRNA previously acylated with a special amino acid is added to the translation system.
- a tRNA acylated with a special amino acid is prepared by conjugating a special amino acid to the 3 ′ end of the isolated orthogonal tRNA using flexizyme in the absence of other tRNA or ARS. Is done.
- a special amino acid is linked to tRNA chemically or enzymatically can be used in principle. The details of the aminoacylation reaction with a special amino acid will be described later.
- Template nucleic acid encoding special peptide in a cell-free translation system, a random amino acid sequence is obtained by performing translational synthesis from a template nucleic acid (mRNA or corresponding DNA) having a random sequence in a region encoding a peptide. Synthesize a peptide library. Therefore, constructing a peptide library includes preparing a library of nucleic acids encoding each peptide and translating it.
- the RNA or DNA sequence encoding this special peptide is designed to encode a linear or cyclic special peptide in which a predetermined special amino acid is introduced at a specified position in a random sequence.
- a predetermined special amino acid is a low molecular weight compound-containing special amino acid.
- the random sequence is designed so that the codon sequence of the template mRNA is such that proteinaceous amino acids appear randomly. You may design so that all 20 types of proteinaceous amino acids may appear, or you may design so that only some proteinaceous amino acids may appear. A modified codon designating this special amino acid is placed at any position of such a random mRNA codon sequence.
- One special amino acid may be introduced into the peptide chain, or two or more may be introduced into the peptide chain.
- NNK N is any base selected from G, A, C, or U, and K is U or G. It is common. However, if this special amino acid is applied to one of the extension codons, such as AUG (as described above, AUG can be used as both the start codon and the extension codon), NNK represents that when a peptide library is constructed with the NNK library In order for AUG to appear randomly as one of the codons, multiple special amino acids are incorporated into the peptide library. That is, when a triplet repeat consisting of an NNK sequence is used as a random sequence, one or more special amino acids encoded by AUG may be placed at unintended positions.
- NNK may be used in addition to NNU and NNC when the merit of making these appear exceeds the demerit of placing the special amino acid at a position other than the desired position.
- a special amino acid other than this special amino acid for example, a special amino acid having a functional group used for cyclization
- these four codons of UGG, CAG, AAG, and GAG can also be used for assignment of special amino acids for circularization.
- An mRNA containing NNU, NNC, or NNK can be obtained by, for example, synthesizing DNA containing NNT, NNC, or NNK using various DNA synthesizers and then transcribing it.
- a cell-free translation system composed of components optimized for the purpose is used by adding a DNA or RNA molecule corresponding to a base sequence serving as a translation template.
- the nucleic acid sequence may additionally contain a base sequence advantageous for translation in accordance with the translation system to be used, in addition to the region encoding the target amino acid sequence, in the same manner as the protein expression system using living cells. it can.
- the efficiency of the translation reaction is increased by including a Shine-Dalgarno (SD) sequence or an epsilon sequence upstream of the initiation codon.
- SD Shine-Dalgarno
- An initiation codon is located at the N-terminus of the region encoding the peptide.
- the start codon is usually the triplet sequence AUG.
- the start codon can be reprogrammed by changing the anticodon sequence to any sequence in the start tRNA synthesized by in vitro transcription reaction, other base sequences can be used as start codons in addition to the AUG codon. it can.
- the special peptide may be cyclized. Therefore, the sequence of RNA or DNA may be designed so that the peptide is circularized using the intramolecular reaction of a linear special peptide synthesized by translation.
- the region encoding the peptide in the base sequence includes the base sequences corresponding to the following (a) to (d) in order along the 5 ′ to 3 ′ direction of the mRNA sequence: (A) a first modified codon that designates a special amino acid having functional group 1 (b) a random sequence comprising a plurality of triplet repeats (c) this special amino acid placed at any position in the random sequence A second modified codon (d) a codon that designates an amino acid having functional group 2.
- the functional group 1 and the functional group 2 are a set of functional groups capable of bond formation reaction, as will be described later.
- the codon specifying the amino acid is a corresponding universal codon
- the codon specifying the amino acid is the third codon. Of modified codons.
- the special amino acid having the functional group 1 for cyclization is an amino acid at the N-terminal of the peptide, and is introduced by a translation initiation reaction with an initiation tRNA.
- a low molecular weight compound-containing special amino acid is introduced by a peptide chain elongation reaction using an elongation tRNA.
- the start tRNA introduces the amino acid linked to the AUG codon at the translation start position into the peptide N-terminus, but the other AUG codon pairs with the extension tRNA having the CAU codon. To do.
- two kinds of amino acids are associated with the AUG codon by two kinds of tRNAs.
- the amino acid having the functional group 2 is a proteinaceous amino acid encoded by a universal codon.
- the amino acid having the functional group 2 is a special amino acid and is encoded by a third modified codon that is an extension codon.
- the third modified codon is other than AUG.
- both the special amino acid having functional group 1 and the amino acid having functional group 2 are extended. It is introduced into the peptide chain by the reaction.
- the first modified codon is other than the starting AUG, and the proteinaceous amino acid having functional group 2 is encoded by a universal codon.
- both the first modified codon and the third modified codon are extension codons, and a sequence other than the starting AUG is assigned.
- the peptide library according to the present invention may be further combined with in vitro display technology so that the peptides constituting the library are accompanied by a nucleic acid sequence encoding the peptide.
- a library in which the phenotype (amino acid sequence of the peptide) is displayed in the genotype (nucleic acid sequence) is constructed.
- a peptide aptamer is selected from a display library in which genetic information is presented (displayed) as a peptide that is a translation product.
- a tag that can be amplified and read by a molecular biological technique is added to each random peptide molecule in the library.
- An in vitro display is a peptide synthesized using a cell-free translation system (also called an in vitro translation system) in association with genetic information.
- Ribosome display, mRNA display, DNA display, etc. are known. It is.
- a RAPID display (see International Publication No. 2011/049157 pamphlet) is also available.
- Each display has a mechanism for associating genetic information recorded in mRNA or DNA with a peptide encoded by the genetic information to associate as a complex of [genetic information]-[translation product].
- ribosome display mRNA, ribosome, and peptide form a complex.
- mRNA display and RAPID display an mRNA-peptide complex is formed.
- DNA display a DNA-peptide complex is formed.
- any in-vitro display library can be used. The in vitro selection, which is a screening method using the in vitro display library, will be described later.
- the RNA or DNA sequence encoding this special peptide may contain a sequence for linking the nucleic acid molecule and its translation product peptide on the 3 ′ end side.
- a linker is usually inserted between the 3 'end of mRNA and puromycin.
- Puromycin functions as a substrate for peptide transfer reaction (aminoacyl-tRNA analog) on the ribosome, and binds the mRNA to the peptide by binding to the C-terminus of the extended peptide.
- the mRNA display method is a technique that integrates genotype and phenotype by linking mRNA and peptide via an appropriate linker in an in vitro translation system. Instead, it is within the purview of those skilled in the art that linkers containing other materials with similar functions can also be used.
- an mRNA-peptide complex library can be formed by hybridization of linker and mRNA in an in vitro translation system, instead of using mRNA with a linker linked in advance.
- a linker for example, a phenylalanine linker (3′-phenylalanine-ACCA-PEG- [base sequence complementary to the 3 ′ end region of mRNA library] -5 ′) prepared using flexizyme is used to connect the mRNA library and the complementary strand.
- an mRNA-peptide complex library is formed (“RAPID display method” described in PCT / JP2010 / 68549).
- a base sequence for hybridization with the linker is included downstream of the region encoding the mRNA peptide (3 'end region).
- a start AUG codon is arranged at the N-terminus of the peptide, and a codon UGC coding for cysteine (Cys) as an amino acid having a functional group 2 is arranged at the C-terminus, followed immediately by a linker.
- a codon encoding GlySerGlySerGlySer follows, a random sequence is formed between the start AUG codon and UGC, and an AUG codon specifying this special amino acid is arranged at the center of the random sequence.
- aminoacyl tRNA necessary for the assignment of special amino acids is prepared by isolating tRNA and aminoacylating in vitro.
- Aminoacylation of an isolated tRNA in vitro means that a desired amino acid is bound to the 3 ′ end of tRNA in the absence of other tRNA or ARS.
- a method applicable to any amino acid is preferable.
- chemical aminoacylation Heckler T. G., Chang L. H., Zama Y., Naka T., Chorghade M. S., Hecht S.
- the most preferable method for aminoacylating tRNA in vitro is a method using ARS ribozyme.
- ARS ribozyme flexizyme developed by the present inventors is preferably used.
- Flexizyme is an RNA catalyst (ARS ribozyme) having a function of acylating an amino acid substrate having a desired structure into an arbitrary tRNA. Unlike the natural ARS protein enzyme, flexizyme has no specificity for each amino acid and each tRNA, and can be aminoacylated using any amino acid other than the amino acid to be originally linked. Specifically, since the ⁇ -position substituent is not included in the amino acid recognition site, it is not limited to L amino acids, but hydroxy acids ( ⁇ -position is a hydroxyl group), ⁇ -N-methylamino acids, ⁇ -N-acylamino acids D-amino acids can also be used as substrates.
- ⁇ -position substituent is not included in the amino acid recognition site, it is not limited to L amino acids, but hydroxy acids ( ⁇ -position is a hydroxyl group), ⁇ -N-methylamino acids, ⁇ -N-acylamino acids D-amino acids can also be used as substrates.
- amino acids that have undergone post-translational modifications such as ⁇ -N-acetyllysine and ⁇ -N-methyllysine can be used as substrates.
- ⁇ -N-acetyllysine and ⁇ -N-methyllysine can be used as substrates.
- WO2008 / 059823 “Translational synthesis of non-naturally-occurring polypeptides and their applications”, Goto et al. “Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides, ACS Chem.
- a special amino acid is introduced into a peptide sequence by adding an orthogonal tRNA acylated with a special amino acid using flexizyme to a cell-free translation system.
- Orthogonal tRNA is not recognized by naturally-occurring ARS (for example, ARS protein enzyme derived from E. coli) inherent in the translation system, so that it is not aminoacylated in the translation system, but is efficient in peptide synthesis reactions on ribosomes. It is a tRNA capable of expressing a designated amino acid by pairing with a codon of mRNA.
- ARS for example, ARS protein enzyme derived from E. coli
- tRNA capable of expressing a designated amino acid by pairing with a codon of mRNA.
- a natural suppressor tRNA derived from a different species or an artificially constructed tRNA is used.
- what is preferably used for the introduction of a special amino acid in the present invention is an artificial tRNA which is an artificial transcription product.
- Flexizyme uses an activated amino acid ester as a substrate, a carbonyl group which is an amino acid reaction point, an aromatic ring which is an amino acid side chain or a leaving group, and a 5′-RCC-3 ′ present at the 3 ′ end of tRNA. Recognizes the sequence portion (R is A or G) and has a catalytic ability to acylate to the 3 ′ terminal adenosine. Flexizyme has no specificity for the anticodon portion of tRNA. In other words, changing the anticodon portion of tRNA to any sequence does not affect the efficiency of aminoacylation. Since flexizyme can link any special amino acid to a tRNA having any anticodon sequence, any special amino acid can correspond to any codon. Therefore, it is possible to prepare a library into which any special amino acid has been introduced.
- RNA sequence The structure (RNA sequence) of a known flexizyme is shown below.
- Prototype Flexizyme Fx [5 ⁇ -GGAUCGAAAGAUUUCCGCAGGCCCGAAAGGGUAUUGGCGUUAGGU-3 ⁇ , 45nt] (SEQ ID NO: 1)
- Dinitrobenzyl flexizyme dFx [5 ⁇ -GGAUCGAAAGAUUUCCGCAUCCCCGAAAGGGUACAUGGCGUUAGGU-3 ⁇ , 46nt]
- SEQ ID NO: 2 Enhanced Flexizyme eFx [5 ⁇ -GGAUCGAAAGAUUUCCGCGGCCCCGAAAGGGGAUUAGCGUUAGGU-3 ⁇ , 45nt] (SEQ ID NO: 3)
- Aminoflexizyme aFx [5 ⁇ -GGAUCGAAAGAUUUCCGCACCCCCGAAAGGGGUAAGUGGCGUUAGGU-3 ⁇ , 47nt] (SEQ ID NO: 4)
- flexizyme skips the process of high-energy intermediate (aminoacylAMP), which is the first step of aminoacylation reaction, and only binds amino acid substrate to tRNA.
- aminoacylAMP high-energy intermediate
- activation of an acyl group can be achieved by esterifying an electron-withdrawing leaving group, but an ester having a very strong electron-withdrawing group is not only hydrolyzed in water but also random RNA.
- Acylation of acetylene occurs simultaneously. Therefore, it is necessary to use an amino acid substrate that is weakly activated so that such a side reaction hardly occurs in a non-catalytic state.
- Such weak activation can be performed using, for example, AMP, cyanomethyl ester, thioester, or benzyl ester having a nitro group, fluorine, or other electron-withdrawing functional group.
- suitable amino acid substrates include aminoacyl-cyanomethyl ester (CME), aminoacyl-dinitrobenzyl ester (DNB: 3,5-dinitrobenzyl ester), or aminoacyl-4-chlorobenzylthioester (CBT: p- chloro-benzyl thioester) and the like, but is not limited thereto.
- CME aminoacyl-cyanomethyl ester
- DNB aminoacyl-dinitrobenzyl ester
- CBT aminoacyl-4-chlorobenzylthioester
- the amino acid substrate must also have an aromatic ring in the amino acid side chain or leaving group so that it can be recognized by flexizyme.
- an amino acid substrate having such an appropriate leaving group as a substrate for flexizyme is sometimes referred to as an activated amino acid ester.
- ⁇ -N-acetyllysine by using ⁇ -N-acetyllysine-CBT as a substrate, eFx and tRNA can be mixed to prepare ⁇ -N-acetyllysine-bound tRNA.
- eFx recognizes the 4-chlorobenzyl group in the leaving group and not the amino acid side chain
- analogs such as ⁇ -N-trifluoroacetyl lysine-CBT and ⁇ -N-thioacetyl lysine-CBT are also available. Similarly, it can be used as a substrate for eFx.
- the acylation reaction with flexizyme may be carried out in a solution or may be carried out using a column using ARS ribozyme immobilized on a carrier.
- a column using ARS ribozyme immobilized on a carrier For example, if the translation reaction scale is as small as 100 ⁇ l or less, tRNA is acylated with flexizyme in the solution, and the pellet obtained by ethanol precipitation of the reaction solution is added to an appropriate buffer (for example, 1 mM potassium acetate, pH 5). Etc.) and added to the translation system.
- an appropriate buffer for example, 1 mM potassium acetate, pH 5). Etc.
- Suitable reaction conditions may be selected as appropriate, but examples of small scale reaction conditions include 0.5-20 ⁇ M tRNA, 0.5-20 ⁇ M flexizyme, 2-10 mM amino acid substrate, 0.6 M A reaction buffer solution containing MgCl 2 at pH 7.5 and 0.1 M may be reacted at 0 ° C. for 1 to 24 hours.
- the translation reaction scale exceeds 100 ⁇ l, it is more convenient to use flexizyme immobilized on a carrier in consideration of reuse of flexizyme.
- the carrier for example, resin, agarose, sepharose, magnetic beads and the like can be used, but are not particularly limited.
- the flexizyme is immobilized on a carrier, for example, Murakami, H., Bonzagni, N. J. and Suga, H. (2002). "Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme.” J. Am. Chem. Soc. 124 (24): 6834-6835. Separation of the reaction product aminoacylated tRNA can be performed by various methods.
- a method of eluting from a column with a buffer containing about 10 mM EDTA As an example, there is a method of eluting from a column with a buffer containing about 10 mM EDTA.
- the resin on which the ARS ribozyme is immobilized can be recycled ten times or more by equilibrating with a reaction buffer, for example.
- tRNA Asn-E2 NNN is an artificial tRNA created by modifying tRNA Asn , which is an elongation reaction tRNA derived from E. coli, and uses various anticodon sequences (NNN, N represents any base). However, if the second modified codon specifying the acetyl lysine analog is AUG, the anticodon sequence is CAU. Since this artificial tRNA is orthogonal to natural ARS, natural amino acids are not linked in the translation system, but are accepted without any problem in the peptide chain elongation reaction on the ribosome.
- the aminoacyl-tRNA in which a special amino acid is linked to this artificial tRNA is combined with an elongation factor (EF-Tu), and is transported to the A site of the ribosome and used in the process of peptide chain elongation.
- tRNA Asn-E2 is an example of an extension tRNA for acylating special amino acids, and it has been confirmed that it can actually be used in the specific cell-free translation system used in the examples. Yes.
- the tRNA for extension reaction that can be used in the present invention is not limited to this.
- the tRNA that can be used for introducing a special amino acid in the peptide chain elongation reaction in the present invention can be appropriately selected according to the components of the cell-free translation system to be used. Let's go. In the present invention, even when an amino acid having a functional group for cyclization is a special amino acid, such a special amino acid is bound to an orthogonal tRNA having an arbitrary anticodon using flexizyme. . In one embodiment of the invention, an amino acid having functional group 1 is placed as the starting amino acid residue. In that case, a functional group for cyclization reaction is introduced into the peptide N-terminal by linking an amino acid having a functional group for cyclization reaction to the start tRNA.
- N ⁇ -chloroacetyl-L (D) -tyrosine which is an L-form or D-form tyrosine having a chloroacetyl group, is linked to the tRNA fMet that is the start tRNA and introduced into the peptide N-terminus. Went.
- the chloroacetyl group introduced into the peptide causes a spontaneous S N 2 reaction with the mercapto group of the cysteine residue inside the peptide, and the peptide is cyclized by a thioether bond (Goto et al., ACS Chem. Biol., 2008, 3, 120-129).
- tyrosine is used as a mother nucleus, but a peptide library can be prepared without any problem even in the L-form and D-form of other 19 kinds of proteinaceous amino acids.
- Initiation tRNA and extension tRNA In natural translation reactions, it is important that the initiation tRNA is used only for initiation of translation and not used in the extension reaction, and conversely, the elongation tRNA is not used in the initiation reaction. Such distinction between the initiation tRNA and the extension tRNA is the same in the present invention.
- artificial tRNA is preferably used for acylating a special amino acid.
- a non-limiting example of an artificial tRNA that is an extended tRNA is tRNA Asn-E2 .
- the base sequence of this tRNA is the natural tRNA Asn of Escherichia coli.
- tRNA Asn- which is a tRNA for elongation reaction that is not subjected to aminoacylation by 20 types of aminoacylation enzymes of E. coli.
- E2 was generated by in vitro transcription.
- NNN corresponds to an anticodon and is changed to correspond to the codon.
- tRNA Asn-E2 5'- GGCUCUGUAGUUCAGUCGGUAGAACGGCGGACU NNN AAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGCCA-3 '( SEQ ID NO:. 5), [locations lost modified, total eight locations s4 U8U, D16U, D20U, t6 A37A, Y39U, m7 G46G, T54U, Y55U. The 34th Q is an anticodon, so it changes according to the codon.] [Mutation sites, total of 4 locations. U1G, C2G, G71C, G72C])
- tRNA fMet One non-limiting example of an artificial tRNA that is a starting tRNA.
- the base sequence of this tRNA is the natural tRNA fMet of E. coli (5′-CGCGGGG s4 UGGAGCAGCCUGGDAGCUCGUCGGGCmU CAU AACCCGAAGAUCGUCGGTYCAAAUCCGGCCCCCGCAACCA-3 ′ (SEQ ID NO: 8)) is the base. (Cm: 2'-O-methylcytidine).
- the present inventors made tRNA fMet , which is a tRNA for initiation reaction in which the modified base was removed and the first C at the 5 ′ end was changed to G from this natural tRNA by in vitro transcription.
- CAU corresponds to the anticodon and corresponds to the start AUG codon.
- TRNA fMet used in the present application 5'-GGCGGGGUGGAGCAGCCUGGUAGCUCGUCGGGCU CAU AACCCGAAGAUCGUCGGUUCAAAUCCGGCCCCCGCAACCA-3 '[SEQ ID NO: 6) [Six U locations with no modification, s4 U8U, D20U, Cm32C, T54U, Y55. points, a total of 1 places .C1G]) important point in the initiator tRNA 5 'end of the first base (natural tRNA fMet in C, is present in the tRNA fMet G), 72 th bases (natural tRNA fMet and present In tRNA fMet , it does not form a complementary strand with A).
- a formyl group is transferred to Met-tRNA fMet by methionylformyltransferase (MTF) (however, there is no meaning when using a special amino acid for initiation such as chloroacetyltryptophan in this part) ), And the binding with EF-Tu is also suppressed.
- MTF methionylformyltransferase
- the peptide library may consist of cyclic peptides.
- the method for cyclizing the peptide is not particularly limited, and for example, the peptide may be cyclized using an intramolecular specific reaction of a non-cyclic peptide synthesized by translation. Cyclization of the peptide is performed by the following steps (i) and (ii).
- a set of functional groups capable of bond-forming reaction means that a bond-forming reaction is possible between the set of functional groups, that is, between functional group 1 and functional group 2, and as a result of the reaction, It is a set of functional groups having an acyclic peptide compound as a cyclic peptide compound.
- Such a set of functional groups is not particularly limited as long as it is a combination of functional groups capable of bond formation reaction.
- there is no particular limitation on the type of reaction between functional groups and various reaction types such as a substitution reaction, an addition reaction, a condensation reaction, and a cyclization addition reaction can be used.
- a set of functional groups for example, —CH 2 —L (L represents a leaving group such as —Cl, —Br and —OSO 2 CH 3 ) and a nucleophilic functional group (—OH, —NH 2 And -SH etc.).
- L represents a leaving group such as —Cl, —Br and —OSO 2 CH 3
- a nucleophilic functional group —OH, —NH 2 And -SH etc.
- An example of the bond formation reaction between the functional group 1 and the functional group 2 is the formation of a cyclic structure by disulfide bonds between two cysteine residues. However, disulfide bonds are easily reduced in vivo. Therefore, in order to form a stable cyclic structure, the bond between the functional group 1 and the functional group 2 is preferably a non-reducing bond.
- An acyclic peptide compound is an acyclic compound included in the special peptide and has the same meaning as a linear peptide.
- the substituent for Ar is not particularly limited, and examples thereof include a hydroxyl group, a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a phenyl group, a phenoxy group, a cyano group, and a nitro group. Is mentioned.
- the structure of the formula (A-3) can be given by a substitution reaction between functional groups.
- the structures of (B-3) and (C-3) can be given by cyclization reaction between functional groups, respectively.
- an acyclic peptide compound is a compound having a set of functional groups on different amino acid units.
- such a non-cyclic peptide compound having a set of functional groups is synthesized by translation synthesis in a cell-free translation system.
- the amino acid having these functional groups for cyclization is not a protein amino acid but a special amino acid, it is introduced into the peptide chain using a genetic code reprogramming technique.
- the translational synthesis of the acyclic peptide compound is (a) a starting tRNA aminoacylated with an amino acid having functional group 1, and (b) an amino acid having functional group 2 and aminoacylated with the amino acid.
- a cell-free translation system comprising at least tRNA, (c) a codon corresponding to the anticodon of the initiating tRNA, and a codon corresponding to the anticodon of tRNA aminoacylated with the amino acid having the functional group 2 at a desired position
- a step of synthesizing an acyclic peptide compound by adding the aminoacylated initiating tRNA of (a) and the mRNA of (c) to the cell-free translation system of (b). Is done by.
- acyclic peptide compound obtained by the method of the first aspect translation starts with a special amino acid having functional group 1, and functional group 2 is present on the proteinaceous amino acid residue introduced in the peptide chain elongation reaction. It will be.
- a cell-free translation system containing no methionine is preferably used.
- the present invention is not limited to this.
- Functional group 1 can be present as a substituent on a carbon atom, such as the ⁇ -carbon and ⁇ -carbon of an amino acid, or can be present on a substituent on such a carbon atom. Also, functional group 1 can be present as a substituent on an amino group nitrogen atom or can be present on a substituent on such an amino group nitrogen atom. The functional group 1 must be capable of forming a bond with the functional group 2.
- the functional group 2 is basically a nucleophilic functional group (—SH, —COOH, —OH, etc.) contained in cysteine, tyrosine and the like.
- a functional group having a group for example, a functional group having a group of —CH 2 —L (L represents a leaving group such as —Cl, —Br, —I and —OSO 2 CH 3 ) is preferable.
- the special amino acid having the functional group 1 is preferably, for example, an amino acid compound having the group (A-1) on the amino group nitrogen atom.
- the amino acid compound include, for example, a compound of the formula (1) Is mentioned.
- R 1 and R 2 represent a hydrogen atom or an arbitrary substituent connected to the ⁇ -position carbon atom by carbon.
- R 1 and R 2 are preferably any one of substituents on the ⁇ -carbon of, for example, 20 types of proteinaceous amino acids.
- R 1 and R 2 are preferably any combination of substituents on the ⁇ -carbon of the proteinaceous amino acid.
- Specific examples of the compound of formula (1) include, for example, formula (1-1): Is mentioned.
- the amino acid having the functional group 2 is introduced in a peptide chain extension reaction in a reconstituted translation system containing at least the amino acid and the corresponding tRNA.
- the functional group 2 is a proteinaceous amino acid
- the proteinaceous amino acid can be introduced into the peptide chain using a codon that designates an aminoacyl tRNA linked to the proteinaceous amino acid without using a modified codon.
- both the amino acid having functional group 1 and the amino acid having functional group 2 are special amino acids.
- Functional group 1 and functional group 2 can be present on a substituent on the amino group nitrogen atom or on a substituent on a carbon atom such as the ⁇ -carbon and ⁇ -carbon.
- Formula (20) to Formula (24) When present on a nitrogen atom, for example, Formula (20) to Formula (24): (Wherein n represents an integer of 1 or more, for example, a number of 1 to 10, and X 1 is as defined above), or as part of the acyl substituent, It can be introduced on a nitrogen atom.
- amino acid compound having the functional group 1 examples include a compound of the formula (2)
- specific examples of the amino acid compound having the functional group 2 include a compound of the formula (3). it can.
- R 1 and R 2 are the same as described above, and Z 1 represents an arbitrary substituent.
- Z 1 include a hydroxyl group, a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a phenyl group, a phenoxy group, a cyano group, and a nitro group.
- Specific examples of the compound of formula (2) include, for example, formula (31): Is mentioned.
- both functional group 1 and functional group 2 are present on amino acid residues introduced in the peptide chain elongation reaction.
- An amino acid having a functional group 1 is a special amino acid and is introduced in a peptide chain elongation reaction using gene reprogramming technology.
- the amino acid having the functional group 2 is a proteinaceous amino acid, and the functional group 2 is basically a nucleophilic functional group (—SH, —COOH and —
- Functional group 1 is a functional group having an appropriate leaving group such as —CH 2 —L (L is a leaving group such as —Cl, —Br, —I, and —OSO 2 CH 3). It is preferably a functional group having a group represented by
- amino acid compound having the functional group 1 examples include a compound of the formula (4).
- m represents an integer of 1 to 10.
- Specific examples of the compound of the formula (4) include a compound in which m is 2, and this compound can be produced, for example, from 2,4-diaminobutyric acid.
- both functional group 1 and functional group 2 are present on the amino acid residue introduced in the peptide chain elongation reaction.
- Both the amino acid having functional group 1 and the amino acid having functional group 2 are special amino acids and are introduced into the peptide chain using genetic code reprogramming technology.
- Functional group 1 and functional group 2 can be present in a substituent on the amino group nitrogen atom or in a substituent on a carbon atom such as ⁇ -carbon and ⁇ -carbon.
- Functional group 1 and functional group 2 are preferably present in substituents on carbon atoms such as ⁇ -carbon and ⁇ -carbon. Examples of the functional group 1 and the functional group 2 include the groups exemplified in the second embodiment.
- amino acids having the functional group 1 include compounds of the formula (5) or the formula (7).
- Z 1 and m are as defined above.
- Specific examples of the compound of formula (7) include, for example, formula (32): Is mentioned.
- amino acid having the functional group 2 examples include a compound of the formula (6) or the formula (8).
- m is as defined above.
- the amino acid compound having the functional group 1 is a compound having the functional group (A-1) (for example, the compound of the above formula (4)), and the amino acid compound having the functional group 2 is homocysteine, mercaptonorvaline, etc. And a combination of a special amino acid having a —SH group.
- a cyclic peptide compound is synthesized by cyclizing the acyclic peptide compound synthesized as described above.
- the conditions for the bond formation reaction between the functional group 1 and the functional group 2 are set according to the type of the functional group set.
- the cyclization of the acyclic peptide compound can be performed by exposing the acyclic peptide compound to appropriate reaction conditions after isolation. Alternatively, cyclization can be performed by adjusting the cell-free translation system to appropriate reaction conditions without isolating the acyclic peptide compound. In addition, depending on the type of functional group, there may be cyclization under the conditions of a cell-free translation system for synthesizing acyclic peptide compounds. In this case, without special adjustment of reaction conditions A cyclic peptide compound can be obtained.
- the reaction conditions for the cyclization of the acyclic peptide compound include, for example, a set of functional groups: —CH 2 —L (L represents a leaving group such as —Cl and —Br) and a nucleophilic functional group.
- -SH for example, after isolation of the acyclic peptide compound, the cell-free translation system can be obtained by heating in an appropriate solvent (eg 40 to 100 ° C.) or without isolation. For example, it can be carried out by keeping at 35 to 40 ° C. for several hours (eg, 37 ° C. for 3 hours).
- the set of functional groups is the set (A)
- the set of functional groups for example, after isolating the acyclic peptide compound, heating in an appropriate solvent (for example, 40 to 100 ° C.) or without isolation
- an appropriate solvent for example, 40 to 100 ° C.
- cell-free translation system at, for example, 35 to 40 ° C. for several hours (eg, 3 hours at 37 ° C.)
- cell-free translation for the synthesis of acyclic peptide compounds when one set of functional groups is the group (A) The reaction of the functional group proceeds in the system and may be isolated as a cyclic peptide compound from a cell-free translation system.
- the acyclic peptide compound isolated from the cell-free translation system is mixed with a monovalent copper salt (copper (II) sulfate ascorbate in an appropriate solvent).
- (B-3) can be obtained by cyclization (Huisgen cyclization).
- the acyclic peptide compound is isolated and then reacted by treatment with potassium ferricyanide (K 3 [Fe (CN) 6 ]) in an appropriate solvent ( C-3).
- a cyclic peptide obtained by translational synthesis of a peptide sequence in which a chloroacetyl group and a cysteine are arranged at both ends is described.
- a peptide having a chloroacetyl group as the functional group 1 is synthesized using a genetic code reprogramming technique.
- the mercapto group spontaneously nucleophilically attacks the chloroacetyl group after translation, and the peptide is cyclized by a thioether bond.
- the peptide is synthesized by adding an initiation tRNA acylated with an amino acid having a chloroacetyl group to a translation system that does not contain methionine.
- the chloroacetyl group can be arranged at a position other than the N-terminus, and in that case, an extended tRNA acylated with an amino acid having a chloroacetyl group is used, and a translation system containing methionine is used.
- the thioether bond formed between the chloroacetyl group and any one cysteine and the remaining cysteines bind to each other.
- a peptide having a plurality of cyclic structures formed with disulfide bonds may be formed.
- Screening method The peptide library produced as described above is useful for screening for selecting the special peptide capable of binding to the target substance.
- One aspect of the screening method includes a step of bringing a peptide library into contact with a target substance, and a step of selecting a peptide that binds to the target substance.
- the target substance is not particularly limited, and may be a low molecular compound, a high molecular compound, a nucleic acid, a peptide, a protein, or the like.
- the target substance can be immobilized on a solid phase carrier and brought into contact with the library of the present invention.
- the “solid phase carrier” is not particularly limited as long as it is a carrier capable of immobilizing a target substance, and is made of a microtiter plate made of glass, metal, resin, etc., substrate, beads, nitrocellulose membrane, nylon membrane , PVDF membranes and the like, and the target substance can be fixed to these solid phase carriers according to a known method.
- the target substance and the library are brought into contact in an appropriately selected buffer solution, and reacted by adjusting pH, temperature, time and the like.
- One aspect of the screening method of the present invention further includes the step of selecting the special peptide bound to the target substance.
- the target substance for example, a peptide that is labeled according to a known method for detectably labeling the peptide, and after the contact step, the surface of the solid phase carrier is washed with a buffer solution, and the peptide bound to the target substance Can be detected.
- Detectable labels include enzymes such as peroxidase and alkaline phosphatase, radioactive substances such as 125 I, 131 I, 35 S, and 3 H, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, near red Examples include fluorescent materials such as outer fluorescent materials, luminescent materials such as luciferase, luciferin, and aequorin, and nanoparticles such as gold colloids and quantum dots.
- an enzyme substrate can be added to cause color development and detection. It can also be detected by binding biotin to the peptide and binding avidin or streptavidin labeled with an enzyme or the like.
- a special peptide library constructed in a cell-free translation system is completely compatible with in vitro display technologies such as mRNA display, and thus has a high diversity of more than 10 12 types. It is possible to create peptide molecules that bind to a target from a special peptide library.
- In-vitro display technology is used as a tool for evolutionary molecular engineering.
- evolutionary molecular engineering for the purpose of creating proteins and peptides with desired functions and properties, potential genes are prepared on a large scale, and clones having the targeted phenotype are selected from them. Basically, a DNA population (DNA library) is first prepared, an RNA population (RNA library) is obtained as an in vitro transcription product, and a peptide population (peptide library) is obtained as an in vitro translation product. From this peptide library, one having a desired function or property is selected by some screening system.
- the peptide population can be poured into a column on which the target protein is immobilized, and a mixture of peptide molecules bound to the column can be recovered.
- a nucleic acid molecule as a template is added like a tag to each peptide molecule by in-vitro display technology.
- mRNA is added to each peptide molecule. Therefore, after collecting the collected peptide-mRNA complex back to DNA with reverse transcriptase and amplifying it with PCR to obtain a biased library containing many clones with the targeted phenotype, the same was repeated. Conduct a selective experiment.
- RNA aptamer in order to avoid the possibility of recovering the RNA aptamer, a reverse transcription reaction can be performed before selection for the purpose of making the nucleic acid portion into a double strand (DNA / RNA hybrid). By repeating this operation, clones having a desired phenotype are enriched in the population as the generation progresses.
- a matching molecule active species
- PCR is performed from the nucleic acid portion of the selected matching molecule.
- the target substance is generally a protein, nucleic acid, carbohydrate, lipid, or any other compound.
- the [genetic information]-[peptide] complex is brought into contact with a target substance, and a complex that presents a peptide bound to the target substance can be selected from many other unbound target substances. It is necessary to separate and recover from the complex by an appropriate method. Many techniques are known as such recovery methods.
- a polyhistidine tag is linked to a target substance and recovered using specific binding of the polyhistidine tag to a carrier on which Ni-NTA is supported.
- specific bindings in addition to these specific bindings, in addition to biotin binding proteins (avidin, streptavidin, etc.) / Biotin combinations, maltose binding protein / maltose, polyhistidine peptides / metal ions (nickel, cobalt, etc.)
- a combination of glutathione-S-transferase / glutathione, antibody / antigen (epitope) and the like can be used, but is not limited thereto.
- a peptide library is brought into contact with a target substance, an active species presenting a peptide bound to the target substance is selected, a nucleic acid sequence of the selected active species is amplified, and the amplified nucleic acid sequence is used as a template. It involves creating a special peptide that binds to a target substance by repeating in vitro selection of selecting active species from a library of peptides synthesized again in a cell-free translation system.
- a specific example of the target substance is an enzyme.
- a library containing peptides having enzyme activity site-directed properties it is possible to obtain peptides having enzyme inhibitory activity as well as simply binding to a target enzyme.
- One aspect of the screening method of the present invention applying in vitro selection was selected by contacting a library with a target substance, selecting a peptide linked with mRNA that binds to the target substance, and reverse transcription.
- a step of synthesizing DNA from mRNA linked to a peptide a step of amplifying the DNA by PCR, obtaining an mRNA library by transcription and binding puromycin to each mRNA, and translating the mRNA in a cell-free translation system
- a step of obtaining a peptide library linked with mRNA and a step of repeating the step of obtaining a peptide library from the contacting step described above one or more times are included. By repeating each step in this manner, peptides having high affinity for the target substance are concentrated.
- the creation of a special peptide compound that binds to the target substance involves collecting the peptide bound to the target substance, analyzing the nucleic acid sequence bound to the peptide, determining the peptide sequence from the nucleic acid sequence, and obtaining the obtained peptide sequence.
- the binding evaluation to the target substance and the inhibition activity can be confirmed, and a special peptide having high activity can be obtained.
- the target substance is an enzyme
- the enzyme inhibitory activity of the obtained peptide can be evaluated to screen for a peptide having the enzyme inhibitory activity.
- the screening method of the present invention when used to select a peptide that binds to the enzyme active site of the target enzyme from a peptide library, the following steps may be performed. (i) preparing a library containing peptides having enzyme active site directivity; (ii) contacting the peptide library with a target substance; and (iii) selecting a peptide molecule that binds to the target substance.
- a peptide having an enzyme inhibitory activity may be selected from a peptide library by further performing the following step (secondary screening step).
- a step of preparing a library containing peptides having an enzyme active site directivity comprising: (ii) contacting the peptide library with a target enzyme molecule; (iii) selecting a peptide molecule that binds to the target enzyme molecule.
- a method for producing a peptide having enzyme inhibitory activity by synthesizing a selected peptide by an appropriate method is also within the scope of the present invention.
- peptides synthesized as such and having enzyme inhibitory activity are within the scope of the present invention.
- Target protein SIRT2 Acetylation of lysine residues, which is a type of post-translational modification of proteins, is dynamically controlled by the action of various acetylases and deacetylases. Sirtuins are one of these deacetylases, and it is known that there are seven humans from SIRT1 to SIRT7.
- sirtuin inhibitor As one type of sirtuin inhibitor, there is a peptidic inhibitor in which only a nearby sequence to be deacetylated from a sirtuin substrate protein is extracted and an acetyl lysine site is converted into an acetyl lysine analog.
- ⁇ -N-trifluoroacetyl lysine Tfa K
- peptides containing them are potent inhibitors of sirtuins4 ) .
- the sequence in the sirtuin substrate protein continues to be used as the peptide sequence surrounding the acetyl lysine analog. Therefore, we constructed a random peptide library with Tfa K and screened to obtain a peptide sequence showing a stronger inhibitory effect.
- an mRNA library as a template is prepared.
- the length of the sequence of the translated portion of mRNA is arbitrary, but five types of lengths of 16 to 20 codons were prepared this time.
- the N-terminus is a start AUG codon (first modified codon), and a codon encoding GlySerGlySerGlySer as a linker is followed by a codon UGC encoding Cys on the C-terminal side.
- a random codon sequence of NNK or NNC or NNU is used between the start AUG codon and UGC. (N represents any one base of A, U, G, and C, and K represents any one base of U, G.) Only one specific codon in this random sequence is introduced with a special amino acid.
- AUG codon (second modified codon).
- peptide library is translated under a modified genetic code table. Specifically, a translation system was constructed in which methionine was removed from 20 normal amino acids, and instead (i) tRNA fMet CAU linked with ⁇ -N-chloroacetylated amino acid, (ii) tRNA Two of AsnE2 CAU linked with Tfa K are prepared and added using flexizyme and translated.
- the tRNA used in (i) is recognized by the initiation factor and paired with the initiation AUG codon
- the tRNA used in (ii) is recognized by the elongation factor and paired with the AUG codon.
- the translated peptide is cyclized by thioether bond between the N-terminal chloroacetyl group and the C-terminal cysteine mercapto group, and a peptide library having trifluoroacetyl lysine in the random sequence is synthesized.
- the C-terminus of is linked to mRNA via Pu (puromycin).
- the peptide library described above is screened by various in vitro display methods such as mRNA display method and ribosome display method, and peptides that bind to SIRT2 are selected. Since a peptide library containing a special amino acid that binds to the target active site is used, the obtained peptide binds to the target active site and inhibits its activity even if screening is performed using only binding as an index. There is a high possibility of doing.
- an inhibitory peptide can be obtained by constructing a peptide library in which it is introduced as a special amino acid.
- a peptide containing N ⁇ -propargyl lysine is known to be an inhibitor of histone demethylase, by screening a peptide library containing this amino acid in the same manner as in the Examples, It is possible to obtain a stronger inhibitor of histone demethylase 5) .
- SIRT2 Since SIRT2 needs to be immobilized on a carrier during mRNA display, it was expressed in E. coli as a construct with a 10xHis tag at the N-terminus and purified using the His tag.
- mRNA represented by the following sequence.
- GGGUUAACUUUAAGAAGGAGAUAUACAU (AUG) (NNK) 1 (NNK) 2 ⁇ ⁇ (NNK) m ( AUG) (NNK) 1 (NNK) 2 ⁇ ⁇ ⁇ (NNK) n (UGC) (GGC) (AGC) (GGC) (AGC ) (GGC) (AGC) (UAG) GACGGGGGGCGGAAA (N represents one of A, U, G, and C, and K represents one of U and G.) (SEQ ID NO: 10)
- mRNA represented by the following sequence.
- GGGUUAACUUUAAGAAGGAGAUAUACAU (AUG) (NNC) 1 (NNC) 2 ⁇ ⁇ (NNC) m (AUG) (NNC) 1 (NNC) 2 ⁇ ⁇ ⁇ (NNC) n (UGC) (GGC) (AGC) (GGC) (AGC ) (GGC) (AGC) (UAG) GACGGGGGGCGGAAA (N represents any one of A, U, G, and C.) (SEQ ID NO: 12)
- the puromycin linker represented by the following sequence was annealed with the above mRNA library and ligated with T4 RNA ligase.
- SPC18 represents PEG with the total number of C and O being 18.
- pdCTCCCGCCCCCCGTCC SPC18 5 CC (Pu) (SEQ ID NO: 13)
- the mRNA linked to the translation linker was translated under the modified genetic code table (FIG. 2).
- a translation system in which methionine was removed from 20 ordinary amino acids was constructed, and instead of (i) tRNA fMet CAU , ⁇ -N-chloroacetyl-L-tyrosine (ClAc- L Y) or ⁇ -N-chloroacetyl-D-tyrosine (ClAc- D Y) linked, (ii) tRNA AsnE2 CAU linked to Tfa K, prepared and added using flexizyme And translated.
- a peptide library containing trifluoroacetyl lysine in a random sequence and cyclized with a thioether bond is synthesized, and Pu is bound to the C-terminal of the peptide, so that the mRNA and the peptide are linked.
- the prepared special cyclic peptide library was mixed with SIRT2 immobilized on TALON beads and stirred at 4 ° C for 30 minutes. The supernatant was removed using a magnet, and the remaining magnetic particles were washed with a buffer. The PCR solution was added to the beads and heated at 95 ° C. for 5 minutes, the peptide was peeled off from the beads, and the supernatant was collected.
- Amplification of sequence information of recovered peptide The peptide-mRNA recovered by binding to SIRT2 was amplified as DNA by reverse transcription and PCR. The obtained DNA was transcribed into mRNA.
- SIRT2 inhibitory activity evaluation of selected peptides The SIRT2 inhibitory activity of selected peptides was examined with an evaluation system using fluorescence. Specifically, first, a peptide having a fluorescent group and a quenching group at both ends of a peptide to be deacetylated to SIRT2 is mixed with SIRT2 to perform deacetylation. Subsequently, when reacted with a protease that cleaves only the deacetylated peptide, only the peptide deacetylated by SIRT2 dissociates the quenching group from the fluorescent group and emits fluorescence.
- NNK mRNA library NNK mRNA libraries, AUG codon of any position in the random sequence is translated into Tfa K, cyclic peptide libraries containing one or more Tfa K is produced.
- ClAc- L Y also in the peptide library using any of ClAc- D Y, the recovery of mRNA is saturated with 4 round. Therefore, when the peptide sequence after 3 rounds was identified, all the sequences had two or more Tfa K (Table 1).
- 1L-01 to 10 are sequentially assigned as SEQ ID NOS: 14 to 23, and 1D-01 to 06 are assigned sequentially as SEQ ID NOS: 24 to 29.
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Abstract
Description
このようにIn vitroディスプレイでは、10の12乗もの多様性を持つペプチドライブラリーをスクリーニングすることが可能であるが、生体の機能を利用してペプチドライブラリーを構築するために、従来はタンパク質性アミノ酸のみから構成されているペプチドライブラリーしか構築されていなかった。この構成要素の低さを乗り越え、阻害能や特異性が十分でない低分子阻害剤をアミノ酸構造中に組み込み、その特殊なアミノ酸を含有するペプチドライブラリーを構築してスクリーニングを行うことができれば、低分子化合物とペプチドそれぞれ単体では達成できないような高い阻害能と選択性を示す阻害剤を獲得することが可能になると期待できる。
遺伝暗号の拡張では、天然の翻訳系でアミノ酸の指定に使われていない終止コドンや人工4塩基コドンを利用し、それらのコドンに特殊アミノ酸を割り当てることで、特殊アミノ酸を含むタンパク質・ペプチドの合成を可能にした。しかし、停止コドンや利用可能な4塩基コドンの数に限りがあるため、同時に利用可能な特殊アミノ酸の数に上限があった(実質3種類以下)。
即ち、本発明は、
〔1〕ランダム配列中の指定された位置に、標的物質に結合しうる部分を有する特殊アミノ酸が配置されたペプチドからなるペプチドライブラリーを翻訳によって製造する方法であって、
(i)ランダムなアミノ酸配列をコードするmRNA配列中に、前記所望の標的物質に結合しうる部分を有する特殊アミノ酸を指定する改変コドンが配置された塩基配列を含むmRNAのライブラリーを調製する工程と、
(ii)改変コドンに指定されるtRNAに前記特殊アミノ酸が連結されたアミノアシルtRNAを調製する工程と、
(iii)前記アミノアシルtRNAを含む無細胞翻訳系で前記mRNAを翻訳して、ランダム配列中に所定の特殊アミノ酸が配置されたペプチドの集合体からなるライブラリーを得る工程と、を含む方法;
〔2〕前記工程(ii)において、前記アミノアシルtRNAが、アシルtRNA合成酵素様活性を持つRNA触媒を用いて、所望の標的物質に結合しうる部分を有する特殊アミノ酸をtRNAに転移することにより調製される、上記〔1〕に記載の方法;
〔3〕前記所望の標的物質に結合しうる部分を有する特殊アミノ酸を指定する改変コドンがAUGコドンであり、mRNAランダム配列がNNC及びNNU(NはA、U、G及びCのいずれか一つの塩基を表す。)のいずれかのトリプレットの繰り返しからなる、上記〔1〕又は〔2〕に記載の方法;
〔4〕前記mRNAランダム配列が、さらにNNK(KはU又はGを表す。)を含む、上記〔3〕に記載の方法;
〔5〕さらに、前記各ペプチドを環状化する工程を含む、上記〔1〕から〔4〕のいずれか1項に記載の方法;
〔6〕前記各ペプチドを環状化する工程は、
前記工程(i)において、前記mRNAランダム配列中に、以下の(A)から(C)のいずれかの組の官能基1及び2をそれぞれ有する2つのアミノ酸をコードする第2及び第3の改変コドンを配置し(但し、官能基2を有するアミノ酸がタンパク質性アミノ酸の場合は、第3の改変コドンに代えて当該タンパク性アミノ酸を指定するコドンを配置してもよい。)、
前記工程(ii)において、第2の改変コドンに指定されるtRNAに前記官能基1を有するアミノ酸を連結したアミノアシルtRNAと、第3の改変コドンに指定されるtRNAに前記官能基2を有するアミノ酸を連結したアミノアシルtRNAとを調製し、これらも用いて前記工程(iii)を行い、
前記工程(iii)の後、前記官能基の間の反応によって環状化することを含む、上記〔5〕に記載の方法
〔7〕前記所望の標的物質に結合しうる部分を有する特殊アミノ酸が低分子化合物含有特殊アミノ酸である、上記〔1〕から〔6〕のいずれか1項に記載の方法;
〔8〕前記標的物質は酵素であり、前記標的物質に結合しうる部分は前記酵素の活性部位に結合すると予測される低分子の基である、上記〔1〕から〔6〕のいずれか1項に記載の方法;
〔9〕前記工程(i)において、さらに、得られたmRNAライブラリーの各mRNAの3´末端に直接、又はリンカーを介してピューロマイシンを結合させる工程を含む、上記〔1〕から〔8〕のいずれか1項に記載の方法;
〔10〕上記〔1〕から〔9〕のいずれか1項に記載の方法で製造されたペプチドライブラリーであって、各ペプチドに、そのペプチドをコードするmRNAが連結されている、ペプチドライブラリー;
〔11〕上記〔1〕から〔9〕のいずれか1項に記載の方法で得られたペプチドライブラリー又は上記〔10〕に記載のペプチドライブラリーを用いて、標的物質に結合しうるペプチドを選択するスクリーニング方法であって、
前記ペプチドライブラリーを標的物質と接触させる工程と、
前記標的物質と結合するペプチドを選択する工程と、
を含む方法:及び
〔12〕上記〔9〕に記載の方法で得られたペプチドライブラリー又は上記〔10〕に記載のペプチドライブラリーを用いて、標的物質に結合しうるペプチドを選択するスクリーニング方法であって、
前記ライブラリーを標的物質と接触させる工程と、
前記標的物質に結合するmRNAが連結されたペプチドを選択する工程と、
逆転写によって、前記選択されたペプチドに連結されたmRNAからDNAを合成する工程と、
前記DNAをPCRで増幅し、転写によってmRNAライブラリーを得て各mRNAにピューロマイシンを結合する工程と、
無細胞翻訳系で前記mRNAを翻訳してmRNAが連結したペプチドライブラリーを得る工程と、
前記接触させる工程からペプチドライブラリーを得る工程を1回以上繰り返す工程と、
を含む方法、
に関する。
また、かかるライブラリーは、翻訳系により合成されるので、多様性を極めて高くすることができ(例えば10の12乗以上)、また、In vitroディスプレイ法と組み合わせれば、標的に対して親和性の高いペプチド配列の濃縮と同定を効率よく行うことも可能である。
また、本発明によれば、例えば、酵素の阻害剤として機能し得る低分子化合物を組み込んだ特殊アミノ酸が導入されたペプチドの集合からなるライブラリーを構築できる。このライブラリーをスクリーニングすることで、単なるアプタマー(結合活性種)ではなく、高い阻害能と選択性を示す阻害剤を獲得することが可能になる
生体の翻訳では、mRNAの3つの塩基の並び(トリプレット)が一つのコドンとして一つのアミノ酸を指定しており、その並びに対応するペプチドが合成される。このとき、コドンとアミノ酸との対応付けは、以下の2段階で行われる。(i) tRNA の末端にアミノアシルtRNA 合成酵素(aminoacyl-tRNA synthetase:ARS)が対応するアミノ酸を連結する。 (ii) tRNA のアンチコドンが対応する mRNA のコドンと対合することにより、mRNA の情報に沿って tRNA 上のアミノ酸が重合されペプチドが合成される。
こうしたコドンとアンチコドンとの対応関係は、ほとんど普遍的に決定されており、64種類のコドンそれぞれに、20種類のアミノ酸のいずれか一つが割り当てられている。普遍遺伝暗号表を以下に示す。
再構成型の翻訳系とは、リボソーム、翻訳因子、tRNA、アミノ酸、およびATPやGTP等のエネルギーソースなど、蛋白質やペプチドの翻訳合成に関わる因子をそれぞれ単離・精製し、混ぜ合わせた翻訳系である。例えば、大腸菌のリボソームを用いる系として次の文献に記載された技術が公知である:H. F. Kung, B. Redfield, B. V. Treadwell, B. Eskin, C. Spears and H. Weissbach (1977) “DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors” The Journal of Biological Chemistry Vol. 252, No. 19, 6889-6894 ; M. C. Gonza, C. Cunningham and R. M. Green (1985) “Isolation and point of action of a factor from Escherichia coli required to reconstruct translation” Proceeding of the National Academy of Sciences of the United States of America Vol. 82, No. 6, 1648-1652 ; M. Y. Pavlov and M. Ehrenberg (1996) “Rate of translation of natural mRNAs in an optimized in vitro system” Archives of Biochemistry and Biophysics Vol. 328, No. 1, 9-16 ; Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa and T. Ueda (2001) “Cell-free translation reconstituted with purified components”Nature Biotechnology Vol. 19, No. 8, 751-755;H. Ohashi, Y. Shimizu, B. W. Ying, and T. Ueda (2007) “Efficient protein selection based on ribosome display system with purified components” Biochemical and Biophysical Research Communications Vol. 352, No. 1, 270-276。
遺伝暗号のリプログラミングには、翻訳系の構成因子を目的に合わせて自由に取り除き、必要な成分だけを再構成してできる翻訳系を利用する。例えば、特定のアミノ酸を除去した翻訳系を再構成すると、当該アミノ酸に対応するコドンが空きコドンになる。続いて、フレキシザイムあるいは化学的アミノアシル化あるいは変異タンパク質酵素を用いたアミノアシル化を利用して、その空きコドンに相補的なアンチコドンを有するtRNAに特殊なアミノ酸を連結し、これを加えて翻訳を行う。これによって、特殊なアミノ酸がそのコドンでコードされることになり、除去したアミノ酸の代わりに特殊なアミノ酸が導入されたペプチドが翻訳される。
ペプチドは環状化すると、(i)プロテアーゼ耐性が向上する、(ii)剛直性が増し膜透過性や標的タンパク質との親和性が向上する、と考えられている。翻訳で生成するペプチドは、2個以上のシステイン残基を含めばジスルフィド結合により環状構造を形成できるが、この結合は生体中で容易に還元されてしまうため、上記のような効果はあまり期待できない。そこで本発明者らは以前に、翻訳された直鎖状のペプチドを非還元性の結合によって環状化させる手法を開発し、報告した(Y. Goto, et al. ACS Chem. Biol. 3 120-129 (2008))。例えば、上記の遺伝暗号のリプログラミング技術により、N末端にクロロアセチル基を有する特殊なペプチドを合成する。このとき、ペプチド中にシステイン残基を配置しておくと、翻訳後に自発的にメルカプト基がクロロアセチル基に求核攻撃し、ペプチドがチオエーテル結合により環状化する。すなわち、クロロアセチル基とメルカプト基という結合形成が可能な一組の官能基をアミノ酸配列に導入することにより、環状化という機能をペプチドに与える。また、このような結合形成が可能な官能基の対は、クロロアセチル基とメルカプト基に限定されない。詳細は後述する。
次に、本発明の実施形態について説明する。
本発明により構築されるペプチドライブラリーは、標的物質に結合しうる部分を有する特殊アミノ酸(以下「本特殊アミノ酸」という。)を所望の位置に有するペプチドの集合体からなることを特徴とする。
ペプチドライブラリーを構成するペプチドの長さは特に限定されないが、例えば、2アミノ酸~25アミノ酸等とすることができる。
本発明において、特殊アミノ酸は、「本特殊アミノ酸」としても用いられ、また、後述する環状化方法にも用いられうる。
これまで、薬剤標的の特定部位、例えば酵素触媒活性部位、に結合する低分子化合物は、一般的にはランダムスクリーニングを経た研究により偶然発見されてきた。しかし、こういった初期低分子化合物が、標的に対し高い親和性や特異性をもつことは極めて稀で、さらなる古典的なメディシナルケミストリー手法であるトライ&エラーによる合成&探索を繰り返す多大な労力を要した。一方、本願発明のように、既知の低分子化合物をペプチド鎖に組み込み、その化合物の周辺に配置されたペプチド配列をランダム化したライブラリーを構築し探索することができれば、低分子化合物を容易に高機能化できる可能性がある。これまでこのようなアプローチにおいては、ライブラリー構築を化学合成に依存せざるを得ず、それ故探索に適応できる多様性(10の6乗程度)に限界があったが、本発明のライブラリーは翻訳系で合成されることから、極めて高い多様性を実現することが可能である。
本発明のライブラリーは、単なるアプタマーではなく阻害活性を有するペプチドを取得するために、酵素活性部位に結合しうる低分子化合物含有特殊アミノ酸を導入したペプチドライブラリーであってもよい。本明細書では、低分子化合物あるいは低分子化合物含有特殊アミノ酸あるいはそのような特殊アミノ酸を含むペプチドが酵素活性部位に結合する性質を、酵素活性部位指向性と表現することもある。
次に、ランダムなアミノ酸配列中の所望の位置に、本特殊アミノ酸が配置されたペプチド(以下、「本特殊ペプチド」と呼ぶ場合もある。)の製造方法を説明する。
本発明のペプチドライブラリーは、
(i)ランダムなアミノ酸配列をコードするmRNA配列中に前記所望の標的物質に結合しうる部分を有するアミノ酸を指定する改変コドンが配置された塩基配列を含むmRNAのライブラリーを調製する工程と、
(ii)改変コドンに指定されるtRNAに前記特殊アミノ酸が連結されたアミノアシルtRNAを調製する工程と、
(iii)前記特殊アミノ酸を連結したtRNAを含む無細胞翻訳系で前記mRNAを翻訳して、ランダム配列中に所定の特殊アミノ酸が配置されたペプチドの集合体からなるライブラリーを得る工程と、を含む方法によって製造される。
工程(i)と工程(ii)の順序は特に問われず、いずれかを先に行っても並行して行ってもよい。
まず、無細胞翻訳系について説明する。
翻訳系とは、ペプチド翻訳合成のための場であり、一般的には方法及びキット(物)の両方を含む概念である。本発明において、特殊ペプチドライブラリーの調製に使用される無細胞翻訳系は、公知の再構成型の翻訳系を用いてもよいし、これをさらに細分化し、より不純物の少ない系を構築して利用してもよい。従来の系と対比させながら、本発明で利用可能なキット(物)としての翻訳系の具体的な構成成分について説明する。
蛋白質類では、翻訳開始因子(例えば、IF1、IF2、IF3)、翻訳伸長因子(例えばEF-Tu、EF-Ts、EF-G)、翻訳終結因子(例えば、RF1、RF2、RF3、RRF)、エネルギーソース再生のための酵素(例えばcreatine kinase, myokinase, pyrophosphatase, nucleotide-diphosphatase kinase)を使用する。この中で、翻訳終結因子・エネルギーソース再生のための酵素の添加は任意である。鋳型DNAからの転写を行うためにT7 RNA polymeraseを加えることもあるが、あらかじめ転写したmRNAを翻訳系に加える場合、RNA polymeraseの添加は不要である。
本発明では、無細胞翻訳系において、ペプチドをコードする領域にランダム配列を持つ鋳型核酸(mRNAもしくは対応するDNA)から翻訳合成を行うことで、ランダムなアミノ酸配列を持つペプチドライブラリーを合成する。したがって、ペプチドライブラリーを構築することは、各ペプチドをコードする核酸からなるライブラリーを調製して、これを翻訳することを含む。
NNU、NNC、NNKを含むmRNAは、例えば各種のDNA合成装置によって、NNT、NNC、NNKを含むDNAを合成し、これを転写することによって得ることができる。
ペプチドをコードする領域のN末端には、開始コドンが配置される。開始コドンは通常はトリプレット配列AUGである。しかしながら、in vitro転写反応により合成された開始tRNAにおいてアンチコドン配列を任意の配列とすることで、開始コドンのリプログラミングが可能であるので、AUGコドンに加えて、他の塩基配列も開始コドンとして利用できる。
そのため、翻訳合成された直鎖状の特殊ペプチドの分子内反応を利用してペプチドが環状化されるように、RNAもしくはDNAの配列を設計してもよい。
例えば、塩基配列においてペプチドをコードする領域が、mRNA配列の5’から3’の向きに沿って、次の(a)から(d)に対応するような塩基配列を順に含む:
(a)官能基1を持つ特殊アミノ酸を指定する、第一の改変コドン
(b)複数のトリプレットの繰り返しからなるランダム配列
(c)ランダム配列中のいずれかの位置に配置された、本特殊アミノ酸を指定する、第二の改変コドン
(d)官能基2を持つアミノ酸を指定するコドン。
官能基2を持つアミノ酸がタンパク質性アミノ酸である場合は該アミノ酸を指定するコドンは対応する普遍コドンであり、官能基2を持つアミノ酸が特殊アミノ酸である場合は該アミノ酸を指定するコドンは第三の改変コドンであることができる。
本発明において、特殊アミノ酸の割り当てに必要なアミノアシルtRNAは、tRNAを単離し、in vitroでアミノアシル化することにより調製される。単離されたtRNAをin vitroでアミノアシル化するとは、他のtRNAやARSが存在しない条件で、所望のアミノ酸をtRNAの3’末端に結合させることを意味する。このようなアミノアシル化の方法としては、どのようなアミノ酸にも適用できる方法が好ましい。このような方法として、例えば、化学的アミノアシル化法(Heckler T. G., Chang L. H., Zama Y., Naka T., Chorghade M. S., Hecht S. M.: T4 RNA ligase mediated preparation of novel “chemically misacylated” tRNAPhe S. Biochemistry 1984, 23:1468-1473.)、または本発明者らが開発したアミノアシルtRNA 合成リボザイム(ARSリボザイム)を用いる方法が公知である。あるいは、適用できるアミノ酸の種類が限られるが、天然のARSを人工的に改変した酵素を用いる方法も利用可能である。
原型のフレキシザイム Fx
[5´-GGAUCGAAAGAUUUCCGCAGGCCCGAAAGGGUAUUGGCGUUAGGU-3´, 45nt](配列番号:1)
ジニトロベンジルフレキシザイム dFx
[5´-GGAUCGAAAGAUUUCCGCAUCCCCGAAAGGGUACAUGGCGUUAGGU-3´,46nt](配列番号:2)
エンハンスドフレキシザイム eFx
[5´-GGAUCGAAAGAUUUCCGCGGCCCCGAAAGGGGAUUAGCGUUAGGU-3´,45nt](配列番号:3)
アミノフレキシザイム aFx
[5´-GGAUCGAAAGAUUUCCGCACCCCCGAAAGGGGUAAGUGGCGUUAGGU-3´,47nt](配列番号:4)
また、本発明では、環状化のための官能基を有するアミノ酸が特殊アミノ酸である場合にも、フレキシザイムを用いてそのような特殊アミノ酸を任意のアンチコドンを持つ直交性(オルソゴナル)tRNAに結合させる。本発明の一態様では、開始アミノ酸残基として官能基1を有するアミノ酸を配置する。その場合、開始tRNAに環状化反応用の官能基を有するアミノ酸を連結することにより、ペプチドN末端に環状化反応用の官能基が導入される。
例えば、後述の実施例では、クロロアセチル基を有するL体もしくはD体のチロシンであるNα-クロロアセチル- L(D)-チロシンを開始tRNAであるtRNAfMetに連結し、ペプチドN末端に導入を行った。ペプチドに導入されたクロロアセチル基は、ペプチド内部のシステイン残基のメルカプト基と自発的なSN2反応を引き起こし、チオエーテル結合によってペプチドが環状化する(Goto et al., ACS Chem. Biol., 2008, 3, 120-129)。この例では、チロシンを母核としているが、他の19種類のタンパク質性アミノ酸のL体及びD体でも問題なくペプチドライブラリーを作製することができる。
天然の翻訳反応において、開始tRNAは翻訳開始のみに用いられ、伸長反応では使用されず、反対に、伸長用tRNAは開始反応には使用されないことは重要である。このような開始tRNAと伸長用tRNAの区別は本願発明においても同様である。
(5’-UCCUCUGs4UAGUUCAGDCGGDAGAACGGCGGACUQUUt6AAYCCGUAUm7GUCACUGGTYCGAGUCCAGUCAGAGGAGCCA-3’(配列番号:7)) がベースになっている(s4U:4-チオウリジン、D:ジヒドロウリジン、Q:キューオシン、t6A:6-スレオニルカルバモイルアデニン、Y:シュードウリジン、m7G:7-メチルグアノシン、T:リボチミジン)。本発明者らは、この天然tRNAに対して、修飾塩基を無くし、かつ変異を導入することで、大腸菌の20種類のアミノアシル化酵素によってアミノアシル化を受けない伸長反応用のtRNAであるtRNAAsn-E2をin vitro転写によって作製した。NNNの箇所がアンチコドンに相当し、コドンに対応するように変化させる。
開始tRNAである人工tRNAの非限定的な一例が、tRNAfMetである。このtRNAの塩基配列は、大腸菌の天然tRNAfMet
(5’-CGCGGGGs4UGGAGCAGCCUGGDAGCUCGUCGGGCmUCAUAACCCGAAGAUCGUCGGTYCAAAUCCGGCCCCCGCAACCA-3’ (配列番号:8))がベースになっている。(Cm:2’-O-メチルシチジン)。本発明者らは、この天然tRNAに対して、修飾塩基を無くし、5’末端最初のCをGに変化させた開始反応用のtRNAであるtRNAfMetをin vitro転写によって作製した。CAUの箇所がアンチコドンに相当し、開始AUGコドンに対応する。(本願で使用したtRNAfMet: 5’-GGCGGGGUGGAGCAGCCUGGUAGCUCGUCGGGCUCAUAACCCGAAGAUCGUCGGUUCAAAUCCGGCCCCCGCAACCA-3’、 (配列番号:6)[修飾を無くした箇所、合計6カ所。s4U8U、D20U、Cm32C、T54U、Y55U。][変異の箇所 、合計1カ所。C1G])開始tRNAにおいて重要な箇所は5’末端の最初の塩基(天然tRNAfMetではC、本願のtRNAfMetではG)が、72番目の塩基(天然tRNAfMet及び本願のtRNAfMetではA)と相補鎖を組まないことである。この非相補鎖によって、メチオニルホルミルトランスフェラーゼ(MTF)によりMet-tRNAfMetにホルミル基が転移されたり(但し、この部分にクロロアセチルトリプトファンのような開始用特殊アミノ酸を利用する場合は、意味は無い)、またEF-Tuとの結合が抑制されたりする。
本発明の一態様において、ペプチドライブラリーは環状ペプチドからなるものであってもよい。ペプチドを環状化する方法は、特に限定されないが、例えば、翻訳合成された非環状のペプチドの分子内特異的反応を利用して環状化してもよい。ペプチドの環状化は、以下の工程(i)及び(ii)により実施される。
(i)結合形成反応が可能な一組の官能基である官能基1及び官能基2を分子内に有する非環状ペプチド化合物を翻訳合成によって合成する工程;及び
(ii)前記官能基1及び官能基2の結合形成反応によって前記非環状ペプチド化合物を環状化する工程。
官能基2がタンパク質性アミノ酸の場合には、改変コドンを用いず、当該タンパク質性アミノ酸を連結したアミノアシルtRNAを指定するコドンを用いて、当該タンパク質性アミノ酸をペプチド鎖に導入することができる。
上述のように製造したペプチドライブラリーは、標的物質に結合しうる本特殊ペプチドを選択するためのスクリーニングに有用である。
スクリーニング方法の一態様は、ペプチドライブラリーと標的物質とを接触させる工程と、前記標的物質と結合するペプチドを選択する工程を含む。
標的物質は、例えば、固相担体に固定して、本発明のライブラリーと接触させることができる。本明細書において、「固相担体」は、標的物質を固定できる担体であれば特に限定されず、ガラス製、金属性、樹脂製等のマイクロタイタープレート、基板、ビーズ、ニトロセルロースメンブレン、ナイロンメンブレン、PVDFメンブレン等が挙げられ、標的物質は、これらの固相担体に公知の方法に従って固定することができる。
標的物質と、ライブラリーは、適宜選択された緩衝液中で接触させ、pH、温度、時間等を調節して反応させる。
本発明において、無細胞翻訳系で構築される特殊ペプチドライブラリーは、mRNAディスプレイを始めとするin vitroディスプレイ技術と完全に適合可能であるため、1012種類以上の高い多様性からなる特殊ペプチドライブラリーから、標的に結合するペプチド分子の創出が可能である。
このように各工程を繰り返すことにより、標的物質に親和性の高いペプチドが濃縮されていく。
(i)酵素活性部位指向性を有するペプチドを含むライブラリーを用意する工程、
(ii)ペプチドライブラリーを標的物質に接触させる工程;及び
(iii)標的物質に結合するペプチド分子を選択する工程。
また、当該方法は、さらに次の工程(二次スクリーニング段階)を行って、ペプチドライブラリーから酵素の阻害活性を有するペプチドを選択してもよい。
(ア)標的酵素に結合するペプチドを選択する一次スクリーニング段階と、(イ)一次スクリーニングで選択されたペプチドの酵素阻害活性を評価し、前記ペプチドが酵素の阻害活性を有するペプチドであることを決定する二次スクリーニング段階を含み、前記一次スクリーニング段階が
(i)酵素活性部位指向性を有するペプチドを含むライブラリーを用意する工程、
(ii)ペプチドライブラリーを標的酵素分子に接触させる工程;
(iii)標的酵素分子に結合するペプチド分子を選択する工程
を含む。
さらに選択されたペプチドを適当な方法で合成することにより、酵素阻害活性を有するペプチドを製造する方法も本発明の範囲内である。また、そのように合成された、酵素の阻害活性を有するペプチドも本発明の範囲内である。
以下では、後述の実施例の項で取り上げるSIRT2を標的とした場合のペプチドライブラリーの構築法とそこからの阻害ペプチド取得法について述べる。(図1)ここで説明される態様はあくまで例示であり、本発明はこの態様に限定されない。
タンパク質翻訳後修飾の一種であるリシン残基のアセチル化は、様々なアセチル化酵素と脱アセチル化酵素の働きによって動的に制御されている。サーチュインは、こうした脱アセチル化酵素の一種で、ヒトにはSIRT1からSIRT7までの7つが存在することが知られている。サーチュインの生体内の働きは完全には解明されていないが、SIRT2については、近年、癌や神経変性疾患との関連が明らかとなり、その阻害剤が注目を集めている1), 2) , 3)。
サーチュイン阻害剤の一種として、サーチュインの基質タンパク質から脱アセチル化される近傍の配列だけを取り出し、アセチルリシン部位をアセチルリシンアナログに変換したペプチド性の阻害剤がある。そうしたアナログの一つであるε-N-トリフルオロアセチルリシン(TfaK)は、サーチュインの活性ポケットに強く結合し、サーチュインに脱トリフルオロアセチル化される速度が脱アセチル化される速度よりもはるかに遅いため、これを含むペプチドはサーチュインの強力な阻害剤となる4)。こうしたペプチド性の阻害剤の開発において、アセチルリシンアナログを囲むペプチド配列はサーチュインの基質タンパク質中の配列が用いられ続けている。
そこで我々は、TfaKを有するランダムペプチドライブラリーを構築し、スクリーニングを行うことで、より強力な阻害効果を示すペプチド配列を獲得することを試みた。
まず、ペプチドライブラリーを翻訳で構築するために、鋳型となるmRNA ライブラリーを調製する。mRNA の翻訳される部分の配列の長さは、任意であるが、今回は16~20コドンの5種類の長さのものを調製した。このうち、N末端は開始AUGコドン(第一の改変コドン)であり、C末端側にはCysをコードするコドンUGCにリンカーとなるGlySerGlySerGlySerをコードするコドンが続く。開始AUGコドンとUGCの間は、NNKもしくはNNCもしくはNNUのランダムなコドン配列とする。(Nは A, U, G, C のいずれか一つの塩基を、K は U, G のいずれか一つの塩基を、それぞれ表す。)このランダム配列中の特定の1コドンだけを、特殊アミノ酸導入用にAUGコドン(第二の改変コドン)とする。
上述のmRNAライブラリーを改変された遺伝暗号表の下で翻訳する。具体的には、通常の20種類のアミノ酸からメチオニンが除去された翻訳系を構築し、代わりに、(i)tRNAfMet CAUにα-N-クロロアセチル化アミノ酸を連結したもの、(ii)tRNAAsnE2 CAUにTfaKを連結したもの、の2つをフレキシザイムを用いて調製・添加して翻訳を行う。ここで、(i)で使用したtRNAは開始因子に認識されて開始AUGコドンに対合するのに対し、(ii)で使用したtRNAは伸長因子に認識されてAUGコドンと対合する。このために、一つのメチオニンというアミノ酸を除去するだけで2つのアミノ酸を導入することが可能である。翻訳されたペプチドは、N末端のクロロアセチル基とC末側のシステインのメルカプト基との間でチオエーテル結合により環状化し、ランダム配列中にトリフルオロアセチルリシンを有するペプチドライブラリーが合成され、さらにペプチドのC末端がPu(ピューロマイシン)を介してmRNAと連結される。
上述したペプチドライブラリーをmRNA ディスプレイ法やリボソームディスプレイ法などの各種in vitro ディスプレイ法によりスクリーニングし、SIRT2に結合するペプチドを選択する。
標的の活性部位に結合する特殊アミノ酸を含有するペプチドライブラリーを用いているので、結合だけを指標にスクリーニングを行っていても、得られたペプチドは標的の活性部位に結合し、その活性を阻害する可能性が高い。
今回の例で取り上げたSIRT2のような脱アセチル化酵素以外にも、様々な酵素に対して活性ポケットに結合して酵素活性を阻害する阻害剤が既知であれば、SIRT2の場合と同様にそれを特殊アミノ酸として導入したペプチドライブラリーを構築して阻害ペプチドを獲得することができる。例えば、Nε-プロパルギルリシンを含有するペプチドは、ヒストン脱メチル化酵素の阻害剤になることが知られているので、実施例と同様にこのアミノ酸を含有するペプチドライブラリーをスクリーニングすることで、より強力なヒストン脱メチル化酵素の阻害ペプチドを得ることが可能である5)。
SIRT2は、mRNA ディスプレイ時に担体に固定する必要があるため、N末端に10xHisタグが付与されたコンストラクトとして大腸菌内で発現し、Hisタグを利用して精製した。
まず、下記の配列を有する二本鎖DNAを調製した。(以下では、Forward 鎖のみを 5´→ 3´ の順で記載する。)
TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACAT(ATG)(NNK)1(NNK)2・・・ (NNK)m (ATG)(NNK)1(NNK)2・・・ (NNK)n(TGC)(GGC)(AGC)(GGC)(AGC)(GGC)(AGC)(TAG) GACGGGGGGCGGAAA(配列番号:9)
(翻訳領域は一つのコドンを一つの( )で括った。Nは A, T, G, C のいずれか一つを、K は T, G のいずれか一つを、それぞれ表す。(m, n)の組み合わせは、 (m, n)= (3, 4), (4, 4), (4, 5), (5, 5), (5, 6)の5種類である。)
GGGUUAACUUUAAGAAGGAGAUAUACAU(AUG)(NNK)1(NNK)2・・(NNK)m(AUG)(NNK)1(NNK)2・・・(NNK)n(UGC)(GGC)(AGC)(GGC)(AGC)(GGC)(AGC)(UAG)GACGGGGGGCGGAAA
(Nは A, U, G, C のいずれか一つを、K は U, G のいずれか一つを、それぞれ表す。)(配列番号:10)
まず、下記の配列を有する二本鎖DNAを調製した。(以下では、Forward 鎖のみを 5´ → 3´ の順で記載する。)
TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACAT(ATG)(NNC)1(NNC)2・・ (NNC)m (ATG)(NNC)1(NNC)2・・ (NNC)n(TGC)(GGC)(AGC)(GGC)(AGC)(GGC)(AGC)(TAG) GACGGGGGGCGGAAA
(翻訳領域は一つのコドンを一つの( )で括った。Nは A, T, G, C のいずれか一つを表す。(m, n)の組み合わせは、 (m, n)= (3, 4), (4, 4), (4, 5), (5, 5), (5, 6)の5種類である。)(配列番号:11)
GGGUUAACUUUAAGAAGGAGAUAUACAU(AUG)(NNC)1(NNC)2・・(NNC)m(AUG)(NNC)1(NNC)2・・・(NNC)n(UGC)(GGC)(AGC)(GGC)(AGC)(GGC)(AGC)(UAG)GACGGGGGGCGGAAA
(Nは A, U, G, C のいずれか一つを表す。)(配列番号:12)
以下の「ピューロマイシンリンカーとの連結」から「回収したペプチドの配列情報の増幅」までのサイクルを繰り返すことで、ランダムなペプチドライブラリーからSIRT2に結合するペプチドを選択した。(図1)
下記の配列で表されるピューロマイシンリンカーを上記の mRNA ライブラリーとアニールさせ、T4 RNA ligase で連結した。(SPC18 はCとOの総数が18であるPEGを表す。)
pdCTCCCGCCCCCCGTCC(SPC18)5CC(Pu)(配列番号:13)
リンカーと連結された mRNA を改変された遺伝暗号表の下で翻訳した(図2)。本実施例の場合には、通常の20種類のアミノ酸からメチオニンが除去された翻訳系を構築し、代わりに、(i)tRNAfMet CAU に α-N-クロロアセチル-L-チロシン(ClAc-LY)またはα-N-クロロアセチル-D-チロシン(ClAc-DY)を連結したもの、(ii)tRNAAsnE2 CAU にTfaKを連結したもの、の2つをフレキシザイムを用いて調製・添加して翻訳を行った。翻訳によって、ランダム配列中にトリフルオロアセチルリシンを含み、チオエーテル結合で環状化したペプチドライブラリーが合成され、ペプチドのC末端に Pu が結合することで、mRNAとペプチドが連結される。
TALONビーズに固定化したSIRT2に、調製した特殊環状ペプチドライブラリーを混合し、4°Cで30分間撹拌した。磁石を利用して上澄みを除去し、残った磁性粒子をバッファーで洗浄した。ビーズにPCR用の溶液を加えて95°Cで5分間加熱し、ペプチドをビーズからはがして上澄みを回収した。
SIRT2に結合して回収されてきたペプチド-mRNAを、逆転写・PCRによってDNAとして増幅した。得られたDNAを転写してmRNAとした。
上記の一連の操作を繰り返し、ペプチド-mRNAの回収率が飽和したところで、増幅されたDNAを用いてTAクローニングを行い、得られたペプチドの配列を同定した。
蛍光を利用した評価系で、選択されたペプチドのSIRT2阻害活性を調べた。具体的にはまず、SIRT2に脱アセチル化されるペプチドの両末端に蛍光基と消光基がついたものをSIRT2と混ぜ、脱アセチル化を行う。続いて、脱アセチル化されたペプチドのみを切断するプロテアーゼと反応させると、SIRT2によって脱アセチル化されたペプチドのみ蛍光基から消光基が解離して蛍光を発する。ここで脱アセチル化反応の際、SIRT2の阻害剤が存在すると、基質ペプチドの脱アセチル化の進行が遅くなり、最終的に得られる蛍光強度が減少する。すなわち、最終的に観測される蛍光の強さによって選択されたペプチドの阻害能が評価できる。
SIRT2の活性ポケットに結合しその活性を阻害するペプチドを獲得するために、配列中に必ず一つ以上のTfaKを含有するペプチドライブラリー(上述のNNK mRNAライブラリーとNNC mRNAライブラリー)を構築し、mRNA ディスプレイ法によって選択を行った。
NNK mRNAライブラリーを翻訳すると、ランダム配列中のいずれかの位置のAUGコドンがTfaKに翻訳され、一つ以上TfaKを含有する環状ペプチドライブラリーが生成する。このペプチドライブラリーを用いてmRNA ディスプレイを行ったところ、ClAc-LY、ClAc-DYのいずれを用いたペプチドライブラリーにおいても、4ラウンド目でmRNAの回収率が飽和した。そこで、3ラウンド後のペプチド配列を同定したところ、全ての配列が2つ以上のTfaKを有していた(表1)。1L-01~10を順に配列番号:14~23とし、1D-01~06を順に配列番号:24~29とする。
NNC mRNAライブラリーを翻訳すると、ランダム配列中のAUGコドンがTfaKに翻訳され、一配列中に一つだけTfaKを含有する環状ペプチドライブラリーが生成する。このペプチドライブラリーを用いてmRNA ディスプレイを行ったところ、ClAc-LYを用いた場合には5ラウンド目で、ClAc-DYを用いた場合には6ラウンド目でmRNAの回収率が飽和した。そこで、それぞれ4、5ラウンド後のペプチド配列の同定を行ったところ、TfaK近傍の配列に高い相同性が見られた(表2)。2L-01~21を順に配列番号:30~50とし、2D-01~19を順に配列番号:51~69とする。
さらに、このうち2つのペプチド(2L-08、2D-08)について、その解離定数Kdおよび阻害定数IC50を決定したところ、いずれのペプチドもKd~3nM、IC50~4 nMという非常に強力な活性を示すことがわかった。
1) T. F. Outeiro, et al. Science 317 516-519 (2007).
2) R. Luthi-Carter et al. Proc. Natl. Acad. Sci. U S A. 107 7927-32 (2010).
3) J. C. Milne and J. M. Denu Curr. Opin. Chem. Biol. 12 11-17 (2008).
4) a) B.C. Simith, and J.M. Denu J. Am. Chem. Soc. 129 5802-5803 (2009). b) B.C. Simith, and J.M. Denu Biochemistry 46 14478-14486 (2009). c) B.C. Simith, and J.M. Denu J. Biol. Chem. 282 37256-37265.
5) Jeffrey C. Culhane, et al. 128 4536-4537 J. Am. Chem. Soc. (2006)
6) Keykavous Parang, et al. 8 37-41 Nat. struct. Biol. (2001)
配列番号2 ジニトロベンジルフレキシザイム dFx
配列番号3 エンハンスドフレキシザイム eFx
配列番号4 アミノフレキシザイム aFx
配列番号5 tRNAAsn-E2
配列番号6 tRNAfMet
配列番号7 大腸菌のtRNAAsn
配列番号8 大腸菌のtRNAfMet
配列番号9 NKK mRNAライブラリーの鋳型DNAの一般配列
配列番号10 NKK mRNAライブラリーの一般配列
配列番号11 NKC mRNAライブラリーの鋳型DNAの一般配列
配列番号12 NKC mRNAライブラリーの一般配列
配列番号13 ピューロマイシンリンカーのDNA部分の塩基配列
配列番号14~29 NNK mRNAライブラリーのメンバーのアミノ酸配列
配列番号30~69 NNC mRNAライブラリーのメンバーのアミノ酸配列
Claims (13)
- ランダム配列中の指定された位置に、標的物質に結合しうる部分を有する特殊アミノ酸が配置されたペプチドからなるペプチドライブラリーを製造する方法であって、
(i)ランダムなアミノ酸配列をコードするmRNA配列中に、前記所望の標的物質に結合しうる部分を有する特殊アミノ酸を指定する改変コドンが配置された塩基配列を含むmRNAのライブラリーを調製する工程と、
(ii)改変コドンに指定されるtRNAに前記特殊アミノ酸が連結したアミノアシルtRNAを調製する工程と、
(iii)前記アミノアシルtRNAを含む無細胞翻訳系で前記mRNAを翻訳して、ランダム配列中に所定の特殊アミノ酸が配置されたペプチドの集合体からなるライブラリーを得る工程と、を含む方法。 - 前記工程(ii)において、前記アミノアシルtRNAが、アシルtRNA合成酵素様活性を持つRNA触媒を用いて、所望の標的物質に結合しうる部分を有する特殊アミノ酸をtRNAに転移することにより調製される、請求項1に記載の方法。
- 前記所望の標的物質に結合しうる部分を有する特殊アミノ酸を指定する改変コドンがAUGコドンであり、mRNAランダム配列がNNC及びNNU(NはA、U、G及びCのいずれか一つの塩基を表す。)のいずれかのトリプレットの繰り返しからなる、請求項1又は2に記載の方法。
- 前記mRNAランダム配列が、さらにNNK(KはU又はGを表す。)を含む、請求項3に記載の方法。
- さらに、前記各ペプチドを環状化する工程を含む、請求項1から4のいずれか1項に記載の方法。
- 前記各ペプチドを環状化する工程は、
前記工程(i)において、前記mRNAランダム配列中に、以下の(A)から(C)のいずれかの組の官能基1及び2をそれぞれ有する2つのアミノ酸をコードする第2及び第3の改変コドンを配置し(但し、官能基2を有するアミノ酸がタンパク質性アミノ酸の場合は、第3の改変コドンに代えて当該タンパク性アミノ酸を指定するコドンを配置してもよい。)、
前記工程(ii)において、前記第2の改変コドンに指定されるtRNAに前記官能基1を有するアミノ酸を連結したアミノアシルtRNAと、第3の改変コドンに指定されるtRNAに前記官能基2を有するアミノ酸を連結したアミノアシルtRNAとを調製し、これらも用いて前記工程(iii)を行い、
前記工程(iii)の後、前記官能基の間の反応によって環状化することを含む、請求項5に記載の方法。
- 前記所望の標的物質に結合しうる部分を有する特殊アミノ酸が低分子化合物含有特殊アミノ酸である、請求項1から6のいずれか1項に記載の方法。
- 前記標的物質は酵素であり、前記標的物質に結合しうる部分は前記酵素の活性部位に結合すると予測される低分子の基である、請求項1から6のいずれか1項に記載の方法。
- 前記工程(i)において、さらに、得られたmRNAライブラリーの各mRNAの3’末端に直接、又はリンカーを介してピューロマイシンを結合させる工程を含む、請求項1から8のいずれか1項に記載の方法。
- 請求項1から9のいずれか1項に記載の方法で製造されたペプチドライブラリーであって、各ペプチドに、そのペプチドをコードするmRNAが連結されている、ペプチドライブラリー。
- 請求項1から9のいずれか1項に記載の方法で得られたペプチドライブラリー又は請求項10に記載のペプチドライブラリーを用いて、標的物質に結合しうるペプチドを選択するスクリーニング方法であって、
前記ペプチドライブラリーを標的物質と接触させる工程と、
前記標的物質と結合するペプチドを選択する工程と、
を含む方法。 - 請求項9に記載の方法で得られたペプチドライブラリー又は請求項10に記載のペプチドライブラリーを用いて、標的物質に結合しうるペプチドを選択するスクリーニング方法であって、
前記ライブラリーと標的物質と接触させる工程と、
前記標的物質に結合するmRNAが連結されたペプチドを選択する工程と、
逆転写によって、前記選択されたペプチドに連結されたmRNAからDNAを合成する工程と、
前記DNAをPCRで増幅し、転写によってmRNAライブラリーを得て各mRNAにピューロマイシンを結合する工程と、
無細胞翻訳系で前記mRNAを翻訳してmRNAが連結したペプチドライブラリーを得る工程と、
前記接触させる工程からペプチドライブラリーを得る工程を1回以上繰り返す工程と、
を含む方法。 - 配列番号:14~69で表されるアミノ酸配列で表されるペプチドのいずれかを含むSIRT2活性阻害剤。
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US10195578B2 (en) | 2019-02-05 |
EP2647720A9 (en) | 2015-04-01 |
JPWO2012074130A1 (ja) | 2014-05-19 |
EP2647720A4 (en) | 2015-05-20 |
EP2647720B1 (en) | 2019-06-19 |
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