Patent Application
20220275534
The content of the ASCII text file of the sequence listing named “20211102_034574_029US1_subseq_ST25” which is 260 kb in size was created on Nov. 2, 2021 and electronically submitted via EFS-Web herewith the application is incorporated herein by reference in its entirety.
The present invention relates to a method of producing a peptide library and a method of producing a peptide-mRNA complex library.
Research and development of various peptide drugs have recently been carried out. Peptide drugs have, as the largest advantage thereof, high affinity and high specificity to target molecules and ability of inhibiting protein-protein interaction which cannot be achieved by low molecular weight compounds. Due to chemical and biological diversity, peptide drugs tend to have higher specificity in interaction with target molecules than that of low molecular weight compounds and as a result, they can have greater physiological activity.
On the other hand, similar to most of other biopharmaceuticals, peptide drugs cannot penetrate a cellular membrane and therefore cannot reach the inside of cells and in addition, compared with large proteins such as antibodies, they are degraded in a short time due to their inferiority in protease resistance. They therefore have a problem with producing a sufficient effect. In recent years, improvement for such problems of peptide drugs has been investigated by subjecting peptides to various modifications.
The present inventors have developed a RAndom Peptide Integrated Discovery (RAPID) system as a protein translation system. This RAPID system is an experiment system that enables construction of a highly-diverse special cyclic peptide library and identification of active peptides from the library by using, in combination, an artificial translation synthesis system specialized in the synthesis of special cyclic peptides and a screening system making use of the characteristics of the translation system or molecular evolution engineering technique.
Since in the RAPID system, an arbitrary amino acid can be linked with an arbitrary tRNA by using an artificial aminoacylated RNA catalyst “flexizyme” (for example, Non-Patent Document 1), a desired amino acid can be bonded to tRNA having a desired anticodon. By making a desired amino acid correspond to an arbitrary codon different from that in a natural genetic code and performing codon reassignment for reprograming a genetic code table, it is possible to create a peptide library having an arbitrary amino acid containing a non-proteinogenic amino acid introduced at an arbitrary position of the peptide or a peptide library having a macrocyclic structure.
The highly-diverse special cyclic peptide library to be constructed includes peptides having enhanced protease resistance, cell permeability, or affinity or specificity to target molecules.
Further, by in vitro selection, peptides which will be active species that bind to an arbitrary target protein can be identified from the resulting library rapidly with high reliability.
Peptides that bind to an arbitrary target protein can be identified rapidly using a peptide library created with a RAPID system, but the fact is that peptides thus identified have still failed to have sufficient cell membrane permeability.
Also for the purpose of creating peptides having activity to a target in cells, there is therefore an eager demand for a method of producing a peptide library including peptides excellent in cell membrane permeability.
An object of the present invention is to provide a novel method of producing a peptide library. In particular, an object is to provide a method of producing a peptide library capable of producing a peptide library including peptides which are expected to have cell membrane permeability and have a highly hydrophobic local structure.
The present inventors have proceeded with investigation to overcome the above-described problem. As a result, it has been found that the above-described problem can be overcome by modifying, in a peptide library, a predetermined amino acid contained in peptides constituting the peptide library with a prenylation enzyme, leading to the completion of the present invention.
The present invention is as follows.
[1] A method of producing a peptide library including prenylated peptide(s), including a step of bringing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.
[2] The method of producing a peptide library as described above in [1], wherein the prenylation enzyme is at least one or more selected from the group consisting of Enzyme 1 to Enzyme 21 and homologs thereof.
[3] The method of producing a peptide library as described above in [1], wherein the prenylation enzyme has any amino acid sequence of the following (1) to (3):
(1) an amino acid sequences represented by any of SEQ ID NOS: 1 to 56,
(2) an amino acid sequence with one or more amino acid deletions, substitutions, or additions in an amino acid sequences represented by any of SEQ ID NOS: 1 to 56 having, and
(3) an amino acid sequence having 80% or more homology with an amino acid sequences represented by any of SEQ ID NOS: 1 to 56.
[4] The method of producing a peptide library as described above in any of [1] to [3], wherein the peptide library includes a cyclic peptide.
[5] The method of producing a peptide library as described above in [4], wherein the cyclic peptide has a cyclic structure formed by bonding of two amino acid residues through a disulfide bond, peptide bond, alkyl bond, alkenyl bond, ester bond, thioester bond, ether bond, thioether bond, phosphonate ether bond, azo bond, C—S—C bond, C—N—C bond, C═N—C bond, amide bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, amine bond, thioamide bond, or triazole bond.
[6] The method of producing a peptide library as described above in any of [1] to [5], including, prior to the prenylation step, a step of translating an mRNA library in a cell-free translation system and preparing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof.
[7] A method of producing a peptide-genotype complex library, including: a step of preparing a peptide-genotype library including a complex between a genotype and a peptide, and a step of bringing the peptide-genotype library into contact with a prenylation enzyme to prenylate at least one Trp or derivative thereof.
[8] The method of producing a peptide-genotype complex library as described above in [7], wherein:
the peptide-genotype library preparation step includes a step of preparing a peptide-mRNA library by mRNA display method; and
the peptide-mRNA library preparation step includes:
a step of bonding a puromycin to a 3′-end of each mRNA of an mRNA library to produce a puromycin-bound mRNA library, and
a step of translating the puromycin-bound mRNA library in a cell-free translation system and preparing a peptide-mRNA library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof.
[9] A screening method for identifying a peptide binding to a target material, including a step of bringing the peptide library prepared by the production method described above in any of [1] to [6] and/or the peptide-genotype complex library prepared by the production method described above in [7] or [8] into a target material, and a step of selecting a peptide binding to the target material.
[10] A method of producing a prenylated peptide, including a step of bringing a peptide having an amino acid sequence containing at least one Trp or derivative thereof into a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.
[11] A method of producing a prenylated peptide as described above in [10], including a step of, prior to the prenylation step, synthesizing a peptide having an amino acid sequence containing at least one Trp or derivative thereof by chemical synthesis.
According to the present invention, a novel method of producing a peptide library can be provided.
(Method of Producing Peptide Library)
The method of producing a peptide library according to the present invention includes a step of bringing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.
The peptide library in the present invention includes at least two peptides. At least one of the peptides included in the peptide library of the present invention has an amino acid sequence containing at least one Trp or derivative thereof.
In the present invention, a peptide library including a highly hydrophobic peptide can be produced by prenylating some of Trps or derivatives thereof with a prenylation enzyme. Since the prenylation enzyme has high substrate tolerance, hydrophobicity of the peptide included in the peptide library can efficiently increase. For the reasons described above, the production method of the present invention makes it possible to efficiently introduce a hydrophobic group into a peptide and thereby produce a peptide library including peptide(s) expected to have cell membrane permeability.
(Step of Preparing Peptide Library)
As the peptide library used in the production method of the present invention, a peptide library may be obtained by production or may be obtained by purchase. When the peptide library is obtained by production, preferred examples of the production method include a method of producing a peptide library by solid-phase or liquid-phase chemical synthesis and a method of translating an mRNA library in a cell-free translation system and thereby preparing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof.
One of preferred aspects of the production method of the present invention includes a step of translating an mRNA library in a cell-free translation system and thereby preparing a peptide library including peptide(s) having an amino acid sequence containing at least one Trp or derivative thereof and a step of bringing the peptide library into contact with a prenylation enzyme and prenylating at least one Trp residue or derivative residue thereof.
The mRNA library contains an mRNA having a plurality of N1N2N3s as a codon.
The term “N1N2N3” as used herein means a codon specifying an arbitrary amino acid and for example, N1, N2, and N3 are each independently selected from adenine (A), guanine (G), cytosine (C), and uracil (U). One mRNA contains a plurality of N1N2N3s and the N1, N2 and N3 are each independently selected. For example, when mRNA has —N1N2N3-N1N2N3-, two N1s, N2s, and N3s may be the same or different from each other.
In the present invention, an arbitrary amino acid is reassigned to N1N2N3. In the reassignment, a codon-amino acid relation different from that in a natural genetic code table can be reassigned or alternatively, the same relation can be reassigned.
The term “natural genetic code table” means a table showing amino acids represented by genetic codes composed of an mRNA triplet in a living body. In the natural genetic code, N1N2N3 represents the following amino acids.
The mRNA library may include an mRNA containing, as the plurality of N1N2N3s, for example, any of a plurality of N1N2Ks, a plurality of N1N2Ss, a plurality of N1N2Ms, a plurality of N1N2Ws, a plurality of N1N2As, a plurality of N1N2Us, a plurality of N1N2Cs, and a plurality of N1N2Gs. In the present specification, N1 and N2 each have the same meaning as described above, Ks each independently represent either uracil (U) or guanine (G), Ss each independently represent either cytosine (C) or guanine (G), Ms each independently represent either adenine (A) or cytosine (C), and Ws each independently represent either adenine (A) or uracil (U).
For convenience, the present invention will next be described using, as an example, an mRNA library including mRNA having a plurality of N1N2Ks, that is, an mRNA library including mRNA having, as a codon, N1N2Ks as N1N2N3. Even if the other mRNA library is used, it may be possible insofar as a translated peptide library contains a peptide to be prenylated. In the natural genetic code table, N1N2K represents 20 kinds of amino acids with G or U in the right column of the table.
In the present specification, for example, Leu may be assigned to UUG as shown in the natural genetic code table or an amino acid other than Leu may be assigned by codon reassignment. Any amino acid can be assigned to a “N1N2K” codon. The term “an amino acid is assigned to a codon” means that a genetic code table is rewritten so that a certain codon encodes the amino acid. The term “an amino acid is assigned to a codon” and the term “a codon is reassigned” have the same meaning in the present specification.
Assignment of an amino acid to each codon different from that of the natural genetic code table is achieved by codon reassignment making use of, for example, an artificial aminoacylated RNA catalyst flexizyme. Using the flexizyme enables bonding of a desired amino acid to a tRNA having an arbitrary anticodon so that an arbitrary amino acid can be assigned to an arbitrary codon. The flexizyme will be described later. In the present invention, “bonding of an amino acid to tRNA” is also expressed by “charging an amino acid with tRNA”, “aminoacylating tRNA”, or “acylating tRNA with an amino acid”.
In the present invention, a non-proteinogenic amino acid may be assigned to “N1N2K”. For example, using, as a non-proteinogenic amino acid, an amino acid having a cyclic structure or an N-alkylamino acid makes it possible to obtain a peptide library having enhanced resistance to proteolysis, cell membrane permeability, and conformational rigidity. Such a peptide library is useful for the screening of peptides targeting an intracellular disease-related molecule or a molecule having a protease activity. When mRNA contains two or more “N1N2Ks”, all of them may be assigned to a non-proteinogenic amino acid or some of them may be assigned to a non-proteinogenic amino acid.
The term “amino acid” as used herein is used in its broadest meaning and it embraces not only natural amino acids but also artificial amino acid mutants and derivatives. The amino acids may be represented by a commonly used single-letter or three-letter code. Examples of the amino acid or derivatives thereof used herein include natural proteinogenic L-amino acids, unnatural amino acids, and chemically synthesized compounds having properties known in the art as characteristics of an amino acid. Examples of the unnatural amino acids include, but not limited to, α,α-disubstituted amino acids (such as α-methylalanine), N-alkyl-α-amino acids, D-amino acids, β-amino acids, and α-hydroxy acids, each having a main chain structure different from that of natural amino acids; amino acids (such as norleucine and homohistidine) having a side-chain structure different from that of natural amino acids; amino acids (such as “homo” amino acids, homophenylalanine, and homohistidine) having extra methylene in the side chain thereof; and amino acids (such as cysteic acid) obtained by substituting the functional group of a carboxylic acid in the side chain of the amino acids by a sulfonic acid group.
The term “amino acid” as used herein embraces proteinogenic amino acids and non-proteinogenic amino acids. The term “proteinogenic amino acid” as used herein means an amino acid (Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr or Val) constituting a protein.
The term “non-proteinogenic amino acid” as used herein means a natural or unnatural amino acid other than the proteinogenic amino acid.
The derivative of Trp is represented, for example, by the following formula (I), formula (II), or formula (III). It is to be noted that the chemical structural formula shown herein embraces tautomers, geometrical isomers, optical isomers, and the like. In particular, asymmetric carbon in the formula (I), formula (II), or formula (III) may have either an R configuration or an S configuration, or a mixture of them.
The ring A in the formula (I) is an aromatic 6-membered ring which may contain a hetero atom; R1 is an arbitrary substituent on the ring A; R2 and R3 each independently represent a hydrogen atom or an arbitrary substituent; m stands for any integer of from 0 to 4, and l stands for an integer of 1 or 2.
Examples of the ring A include benzene ring, pyridine ring, pyridazine ring, pyrimidine ring, and pyrazine ring.
Although the substituents R1, R2, and R3 are not particularly limited, examples include a hydroxy group; halogen groups such as fluorine group, chloro group, bromo group, and iodo group; alkyl groups having from 1 to 10 carbon atoms such as methyl group, ethyl group, and propyl group; aryl groups such as phenyl group and naphthyl group; aralkyl groups such as benzyl group; and aralkyloxy groups such as benzyloxy group.
In the formula (II), the ring A, R1, R2, m, and l have the same meanings as the ring A, R1, R2, m, and l in the formula (I). The ring A in the formula (II) is preferably a benzene ring.
In the formula (III), the ring A, R1, R2, m, and l have the same meanings as the ring A, R1, R2, m, and l in the formula (I). The ring A in the formula (III) is preferably a benzene ring.
Examples of the Trp derivative represented by the formula (I) include the following derivatives.
In the above derivatives, R1, R2, R3 and m have the same meanings as R1, R2, R3, and m in the formula (I).
Specific examples of the derivatives of Trp include D-tryptophan, 5-methyltryptophan, 5-hydroxytryptophan, 7-azatryptophan, and 5-benzyloxytryptophan.
It is presumed that since the Trp residue is prenylated to form a structure having an alkylated main-chain nitrogen as shown in the following formula (T1), a hydrogen bond donor decreases and the peptide containing the Trp residue tends to have improved hydrophobicity and membrane permeability (Refer to Beilstein J Org Chem. 2017 Feb. 22; 13:338-346.).
Here, the prenyl group is a structural unit composed of C5 isoprene unit(s) and it is the following structure portion when the formula (T1) is used as an example.
(In the above structure, n stands for any integer of from 0 to 11).
Although the steric configuration in the formula (T1) is not particularly limited, examples include steric configurations represented by the following formula (T1-1) and (T1-2). The steric configuration in the formula (T1) preferably has the steric configuration represented by the following formula (T1-1).
When the derivative residue of Trp is prenylated, the resulting product can be represented by the following formulas (I-1), (II-1), and (III-1).
(In the formulas (I-1), (II-1), and (III-1), the ring A, R1, R2, R3, m, and l have the same meanings as the ring A, R1, R2, R3, m, and l in the formula (I), respectively).
By the method of producing a peptide library according to the present invention, a peptide library including several kinds of peptides including an amino acid encoded by “N1N2N3” can be produced. The number of amino acids encoded by “N1N2N3” included in each peptide is not particularly limited, it can be set at, for example, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30. The position of an amino acid encoded by “N1N2N3” in each peptide is not particularly limited. Amino acids encoded by “N1N2N3” in each peptide may be adjacent to each other or may be separated from each other.
In the method of producing a peptide library according to the present invention, an mRNA library including mRNA(s) which encode each peptide of the peptide library, respectively, and each of which contains at least one N1N2N3 is prepared.
The sequence of mRNAs which encode the peptides of the peptide library, respectively, can be determined depending on the amino acid sequence of the peptides constituting the peptide library and such an mRNA library can be prepared by synthesizing a DNA library encoding it and transcribing the resulting library.
For example, as shown below in Table 2, an mRNA library including mRNAs having, as N1N2N3, NNG, NNK, NNS, NNM, NNW, NNA, NNU or NNC can be designed while making use of the natural genetic code table.
In Table 2, the Ns are each independently selected from adenine (A), guanine (G), cytosine (C), and uracil (U).
In the above table, Ks each independently represent either uracil (U) or guanine (G);
Ss each independently represent either cytosine (C) or guanine (G);
Ms each independently represent either adenine (A) or cytosine (C); and
Ws each independently represent either adenine (A) or uracil (U).
In the above table, n is an arbitrary integer of 1 or more, preferably any integer of from 1 to 100, more preferably any integer of from 3 to 50, still more preferably any integer of from 3 to 20, still more preferably any integer of from 6 to 15.
When the natural genetic code table is used, a triplet encoding Trp is only one kind, that is, UGG, among 64 kinds of N1N2N3s. In theory, this suggests that the probability of one N1N2N3 triplet encoding Trp is 1 in 64. On the other hand, for example, the probability of one NNK triplet encoding Trp is 1 in 32. An mRNA library including, for example, mRNA(s) having 12 triplet repeats and having NNK as N1N2N3 can be designed while making use of the natural genetic code table. Such an mRNA library theoretically has diversity as high as 1.15×1018 and includes 22% mRNAs not only containing no stop codon but also containing at least one UGG.
For example, the probability of one NNG triplet encoding Trp is 1 in 16. Such an mRNA library including NNG triplets and having n of 12 theoretically has diversity as high as 2.8×1014 and includes 26% mRNAs not only containing no stop codon but also containing at least one UGG.
The present inventors have found that in the prenylation of a Trp residue with a prenylation enzyme, an amino acid residue upstream or downstream of the Trp residue has an influence on a modification efficiency. By using an mRNA library including mRNA(s) containing a codon encoding an amino acid residue which is adjacent to a Trp or derivative thereof to be prenylated and promotes the prenylation of the Trp or derivative thereof, the prenylation efficiency of the Trp or derivative thereof can be improved.
For example, as shown below in Table 3-1, using an mRNA containing UGG and a codon (XXX, YYY) adjacent to UGG and encoding an amino acid residue that promotes prenylation enables efficient introduction of a prenyl group in a peptide library.
In Table 3-1, XXX is a degenerate codon sequence that specifies a group of upstream amino acids which become more susceptible to a prenylation enzyme used.
YYY is a degenerate codon sequence that specifies a group of downstream amino acids which become more susceptible to a prenylation enzyme used.
In the above table, n and m each independently represent an any integer of 0 or more and the sum of n and m is preferably any integer of from 1 to 100, more preferably any integer of from 3 to 50, still more preferably any integer of from 3 to 20, still more preferably any integer of from 4 to 13.
N1N2N3 has the same meaning as defined above and is, for example, a codon selected from the group consisting of NNK, NNS, NNW, NNM, NNA, NNC, NNG, and NNU. Two or more N1N2N3s are each a codon independently selected.
A modification efficiency with a prenylation enzyme changes depending on the kind of a prenylation enzyme used and it can be enhanced by adjusting an upstream and/or downstream amino acid residue adjacent to Trp (XXX, YYY as a corresponding codon). The amino acid sequence(s) for efficiency enhancement composed of Trp and the upstream and/or downstream amino acid residue(s) adjacent to Trp, XXX and YYY can be determined as needed by comprehensively preparing peptides in a cell-free translation system and carrying out an efficiency confirmation test by performing in vitro prenylation with a predetermined prenylation enzyme.
When KgpF is used as a prenylation enzyme, prenylated Trp can be introduced into a peptide efficiently by being contained the mRNAs shown in Table 3-2, among mRNAs in Table 3-1, in a library.
In Table 3-2, XXX, NNK, n, and m have the same meanings as described above.
Using an mRNA library including such a mRNA as the mRNA shown in Table 3-2, having an n and m of arbitrary integer and containing UGG while making use of the natural genetic code table makes it possible to fix the appearance position of UGG in the resulting peptide library and control the position of the Trp residue to be prenylated in the peptide. Further, when, for example, KgpF is used as a prenylation enzyme, the prenylation efficiency of the Trp residue can be made higher by designating the triplet XXX upstream of UGG to RYU or CMG and/or designating the triplet downstream of UGG to NNK. Such an mRNA library theoretically has diversity as high as 6.8×1016 and includes 73% mRNAs not only containing no stop codon but also containing at least one UGG.
In Table 3-2, when XXX represents RYU or CMG, Rs each independently represent G or A, Ys each independently represent U or C, Ms each independently represent A or C, and Ks each independently represent G or U.
In the method of producing a peptide library according to the present invention, the mRNAs of the mRNA library are each translated in a cell-free translation system having, for example, 20 kinds of tRNAs having an anticodon for any of 20 N1N2N3 codons and charged with an amino acid corresponding to the codon.
For codon reassignment, a translation system obtained by arbitrarily removing a component constituting a translation system depending on a purpose and then reconstituting with only necessary components can be used. For example, when a translation system from which a specific amino acid has been removed is reconstituted, a codon corresponding to the amino acid becomes an empty codon, that is, a codon not encoding any amino acid. An arbitrary amino acid is then linked to a tRNA having an anticodon complementary to the empty codon by making use of flexizyme or the like. After addition of the resulting tRNA, translation is performed. The arbitrary amino acid is then encoded by the codon and a peptide having the arbitrary amino acid introduced therein instead of the removed amino acid is translated.
The tRNA to be used in the present invention may be a wild-type Escherichia-coli derived tRNA or an artificial tRNA prepared by in vitro transcription.
In the present invention, 20 kinds of tRNAs corresponding to 20 kinds of N1N2N3 to be used in the translation system may have the same sequence except for an anticodon loop portion. Using such a constitution enables the tRNAs to have uniform reactivity without enhancing or reducing the reactivity of a specific tRNA, leading to expression of a predetermined peptide with good reproducibility.
The term “cell-free translation system” as used herein means a translation system not containing cells. As the cell-free translation system, for example, an Escherichia coli extract, a wheat germ extract, a rabbit reticulocyte extract, or an insect cell extract can be used. Also usable is a reconstituted cell-free translation system constructed by the reconstitution of ribosome protein, aminoacyl-tRNA synthetase (aaRS), ribosomal RNA, amino acid, rRNA, GTP, ATP, translation initiation factor (IF), elongation factor (EF), release factor (RF), and ribosome regeneration factor (RRF), and another factor necessary for translation, each of which has been purified.
The system may contain an RNA polymerase for simultaneously performing transcription from DNA. As a commercially available cell-free translation system, usable are Escherichia-coli derived systems such as RTS-100 (registered trademark) of Roche Diagnostics, reconstituted translation systems such as PURESYSTEM (registered trademark) of PGI, PUREfrex of GeneFrontier, PURExpress In Vitro Protein Synthesis Kit of New England Biolabs, and systems using a wheat germ extract such as those of ZOEGENE and CellFree Sciences.
As a system using a ribosome of Escherichia coli, for example, techniques described in the following documents are known: H. F. Kung et al., 1977. The Journal of Biological Chemistry Vol. 252, No. 19, 6889-6894; M. C. Gonza et al., 1985, Proceeding of National Academy of Sciences of the United States of America Vol. 82, 1648-1652; M. Y. Pavlov and M. Ehrenberg, 1996, Archives of Biochemistry and Biophysics Vol. 328, No. 1, 9-16; Y. Shimizu et al., 2001, Nature Biotechnology Vol. 19, No. 8, 751-755; H. Ohashi et al., 2007, Biochemical and Biophysical Research Communications Vol. 352, No. 1, 270-276.
By using the cell-free translation system, a highly-pure expression product can be obtained without purification.
The cell-free translation system of the present invention may be used for not only for translation but also for transcription after addition of factor(s) necessary for transcription.
In the present invention, it is preferred not to add a natural aminoacyl-tRNA synthetase corresponding to an N1N2K codon to the cell-free translation system. In this case, the tRNA corresponding to the N1N2K codon can be charged with a corresponding amino acid, for example, by an artificial aminoacyl-tRNA synthetase such as flexizyme.
The peptide library in the present invention may include a cyclic peptide obtained by cyclization of a peptide. The term “cyclic peptide” as used herein means a peptide having a cyclic structure composed of 4 or more amino acids. The term “cyclic structure” means, in a linear peptide, a closed ring structure formed in the molecule by bonding, directly or via a linker or the like, of two amino acids separated from each other by two or more amino acid residues. The term “separated from each other by two or more amino acid residues” means that the two amino acids have at least two amino acid residues therebetween.
The cyclic structure is formed by bonding of two amino acids through a disulfide bond, peptide bond, alkyl bond, alkenyl bond, ester bond, thioester bond, ether bond, thioether bond, phosphonate ether bond, azo bond, C—S—C bond, C—N—C bond, C═N—C bond, amide bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, amine bond, thioamide bond, triazole bond, or the like. The kind of the bond is however not limited to them.
Cyclization of a peptide is sometimes useful for stabilizing the structure of the peptide and enhancing its affinity for a target.
The number of amino acids constituting a cyclic structure, in other words, amino acids forming a ring in the cyclic peptides is not particularly limited insofar they are four amino acids or more. They may be, for example, 4 amino acids or more, 5 amino acids or more, 8 amino acids or more, 15 amino acids or less, 20 amino acids or less, 25 amino acids or less, 30 amino acids or less, or the like.
The cyclization is not limited to that formed by bonding of the N-terminal and C-terminal amino acids of a peptide, but it may be formed by bonding of a terminal amino acid and a non-terminal amino acid or bonding of non-terminal amino acids. When one of the amino acids bonded for ring formation is a terminal amino acid and the other one is a non-terminal amino acid, the resulting cyclic peptide has a cyclic structure having a linear peptide attached thereto like a tail. Such a structure is sometimes called “lasso type” herein.
The term “amino acids adjacent to each other” as used herein means amino acids adjacent to each other even after a ring is formed. Amino acids adjacent to each other are, in a peptide, amino acids which are not separated from each other by one or more amino acid residues and in which amino acid residues have bonded to each other directly.
For cyclization, for example, a chloroacetylated amino acid may be used as the N terminal and a Cys may be placed downstream (on the C-terminal side) of the N terminal. In this case, through thioether bonding between the N-terminal amino acid and the Cys placed downstream (on the C terminal side) of the N terminal, spontaneous cyclization of the peptide occurs after expression. A thioether bond formed between a chloroacetylated amino acid and a Cys is not susceptible to degradation even under reducing conditions in a living body so that a physiologically active effect can be continued while prolonging the half-life in blood of the peptide.
Examples of the chloroacetylated amino acid include N-chloroacetyl-L-alanine, N-chloroacetyl-L-phenylalanine, N-chloroacetyl-L-tyrosine, N-chloroacetyl-L-tryptophan, N-3-chloromethylbenzoyl-L-phenylalanine, N-3-chloromethylbenzoyl-L-tyrosine, N-3-chloromethylbenzoyl-L-tryptophane, N-3-(2-chloroacetamido)benzoyl-L-phenylalanine, N-3-(2-chloroacetamido)benzoyl-L-tyrosine, and N-3-(2-chloroacetamido)benzoyl-L-tryptophane, and D-amino acid derivatives corresponding thereto.
Using, for example, Nγ-(2-chloroacetyl)-α,γ-diaminobutyric acid or Nγ-(2-chloroacetyl)-α,γ-diaminopropanoic acid as the chloroacetylated amino acid enables introduction of itself into any site of the peptide chain so that a thioether bond is formed between the amino acid at any position and a cysteine in the same peptide to form a cyclic structure.
The cyclization method may be carried out according to the method described, for example, in Kawakami, T. et al., Nature Chemical Biology 5, 888-890 (2009); Yamagishi, Y. et al., Chem Bio Chem 10, 1469-1472 (2009); Sako, Y. et al., Journal of American Chemical Society 130, 7932-7934 (2008); Goto, Y. et al., ACS Chemical Biology 3, 120-129 (2008); and Kawakami T. et al, Chemistry & Biology 15, 32-42 (2008).
The chloroacetylated amino acid and Cys may be bound to the peptide of the present invention directly or may be bonded via a linker or the like.
When the C-terminal amino acid of the cyclic peptide is not used for cyclization, this C terminal is not limited to a carboxyl group or a carboxylate group but may also be in the form of an amide or ester. The cyclic peptide of the present invention embraces salts of the cyclic peptide. Examples of the salts of the cyclic peptide include salts with a physiological acceptable base or acid such as addition salts of an inorganic acid (such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, or phosphoric acid), addition salts of an organic acid (such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, or acetic acid), inorganic bases (such as ammonium hydroxide, alkali or alkaline earth metal hydroxides, carbonates, or bicarbonates), and addition salts of an amino acid.
The cyclic peptide of the present invention may be those modified through phosphorylation, methylation, acetylation, adenylylation, ADP ribosylation, glylcosylation or the like insofar as it solves the problem of the present invention. It may be those fused with another peptide or protein.
(Prenylation Step)
The method of producing a peptide library according to the present invention includes a step of bringing a peptide library into contact with a prenylation enzyme and prenylating at least a part of Trps or derivatives thereof included in at least some of peptides. The term “prenylation enzyme” as used herein means an enzyme capable of bonding a prenyl group composed of a C5 isoprene unit to at least a part of Trps or derivatives thereof contained in the peptide. The term “prenyl group” as used herein embraces a group having a plurality of isoprene units consecutively bonded to each other. Prenyl groups having 1, 2, 3, 4, 5, 6, 8, and 10 isoprene units may be called “dimethylallyl group”, “geranyl group”, “farnesyl group”, geranylgeranyl group”, “geranylfarnesyl group” “hexaprenyl group”, “octaprenyl group”, and “decaprenyl group”, respectively.
In the prenylation step in the method of producing a peptide library according to the present invention, a peptide library is preferably brought into contact with a prenylation enzyme in the presence of a donor for a prenyl group. The donor for providing a prenyl group is not particularly limited and examples include dimethylallyl diphosphate (DMAPP), isoprenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and phytyl diphosphate (PDP). These prenyl group donors may be used either singly or in combination of two or more.
The prenylation step in the method of producing a peptide library in the present invention may be performed in the presence or absence of a cofactor. The term “cofactor” as used herein means a material capable of enhancing the catalytic activity of an enzyme. Although the cofactor is not particularly limited, examples include magnesium ion, copper ion, iron ion, manganese ion, molybdenum ion, nickel ion, selenium ion, zinc ion and the like. They may be used either singly or in combination of two or more. Of these, magnesium ion is preferred.
Whether the cofactor is used or not in the method of producing a peptide library according to the present invention may be selected as needed, depending on the prenylation enzyme used. When a peptide library to be subject to prenylation is prepared in a cell-free translation system, prenylation can be performed without adding a cofactor because cofactor(s) is sometimes contained in this cell-free translation system.
Specific examples of the prenylation enzyme usable in the present invention include KgpF, OltF, TruF1, TruF2, PatF, LynF, AcyF, OscF and PagF, and homologs thereof capable of prenylating a Trp residue or Trp derivative, each listed below in Tables 4-1 to 4-2. These prenylation enzymes can modify a very wide range of substrates regardless of the kind of amino acid residues adjacent to a Trp or derivative thereof to be prenylated, as verified in Examples described later. The term “amino acid derivative” as used herein is used in its broadest sense. It embraces any amino acids other than natural amino acids and also includes the above-described artificial amino acid mutants and derivatives. The prenylation enzyme in the present invention can prenylate even an amino acid residue contained in a cyclic peptide so that also from this standpoint, it can modify a very wide range of substrates.
The prenylation enzyme can also be used while being supported on a solid-phase support such as magnetic beads. The prenylation enzyme in the present invention therefore embraces an aspect of an enzyme supported on a solid-phase support. The prenylation enzyme in the present invention may have an addition sequence of His tag, glutathione-S-transferase (GST), maltose-bound protein (MBP), or the like.
The prenylation enzyme and homologs thereof capable of prenylating a Trp or derivative thereof are not particularly limited insofar as they can prenylate a Trp or derivative thereof. As the prenylation enzyme, preferred is, as a result of selecting a known prenylation enzyme (enzyme obtained as a result of BLAST search analysis), forming a sequence alignment by using, for example, a Clusta IW, MEGA7 software, and performing phylogenetic analysis, an enzyme having an amino acid sequence found to have homology with the amino acid sequence of this known prenylation enzyme. The enzyme having an amino acid sequence found to have homology as a result of analysis includes a protein annotated as hypothetical protein.
As the prenylation enzyme, for example, Enzyme 1 to Enzyme 21 listed in Table 4-1 and Table 4-2 can be used. These enzymes may be used in combination of two or more.
Homologues of the prenylation enzymes from Enzyme 1 to Enzyme 21 are not particularly limited insofar as they are enzymes serving to prenylate a Trp or derivative thereof and they may be enzymes having an amino acid sequence having 20% or more homology with the alignment of Enzyme 1 to Enzyme 21.
The prenylation enzyme in the present specification is not particularly limited insofar as it has at least a domain engaged in a prenylation reaction. The homologs of Enzyme 1 to Enzyme 21 may be an aspect of an enzyme having a domain engaged in the prenylation reaction and a domain engaged in the other function (such an enzyme may also be called “bi-functional enzyme”) or may be an aspect of two or more enzymes each having a domain engaged in prenylation.
Of the enzymes listed in Table 4-1 and Table 4-2, enzymes of Group 1 and Group 2 are preferred, with enzymes of Group 1 being more preferred.
Described specifically, the above prenylation enzyme is preferably composed of any of the following amino acid sequences.
(1) An amino acid sequences represented by any of SEQ ID NOS. 1 to 56 listed in the following Table 4-3 to Table 4-8.
(2) An amino acid sequence with one or more amino acid deletions, substitutions or additions in an amino acid sequences represented by any of SEQ ID NOS. 1 to 56.
(3) An amino acid sequence having preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, particularly preferably 98% or more homology with an amino acid sequences represented by any of SEQ ID NOS. 1 to 56.
nigro-viridis]
paucivesiculata
Didemni
The term “with one or more amino acid deletions, substitutions or additions” as used herein means that the number of amino acids deleted, substituted or the like is not particularly limited insofar as a prenylation enzyme thus obtained retains a function of prenylating an amino acid residue. In this sentence, the term “more” means an integer of 2 or more, preferably several, for example, from 2 to 15, more preferably from 2 to 10, still more preferably from 2 to 5, still more preferably 2, 3, or 4. The position of deletion, substitution or addition in each prenylation enzyme may be any of N terminal, C terminal and therebetween in each prenylation enzyme insofar as the resulting prenylation enzyme retains a function of prenylating an amino acid residue.
The term “having Y % or more homology with an amino acid sequence represented by SEQ ID NO: X” as used herein means that when respective amino acid sequences of two polypeptides are aligned to have the maximum agreement, a proportion of the number of amino acid residues common to them to the total number of amino acids shown by SEQ ID NO: X is Y % or more.
The prenylation enzyme is capable of prenylating a peptide containing a motif of, for example but not particularly limited to, X2X1X3 or X4X2X1X3X5 (X1 represents W which is an amino acid residue or derivative thereof to be prenylated, X2, X3, X4, and X5 each independently represent an arbitrary amino acid residue. The arbitrary amino acid residue also includes ClAc-D-Tyr). Although the amino acid residue adjacent to X1 including X2, X3, X4 and X5 is not particularly limited if the prenylation enzyme of the present invention is used, it is preferably an amino acid residue promoting prenylation of the amino acid residue X1 to be prenylated from the standpoint of improving the reaction efficiency of prenylation.
X1 is W or a derivative thereof.
The following X2 to X5 and the following motifs are shown as examples when KgpF is used as a prenylation enzyme. When a prenylation enzyme other than KgpF is used, preferable X2 to X5 and the motif can be set by, as described above, comprehensively preparing peptides in a free-cell translation system and performing an efficiency confirmation test by in vitro prenylation with a predetermined prenylation enzyme.
X2 is preferably V, T, I, A, P, Q, N, S, F, H, W, L, or G, or a derivative of any of them, more preferably V, T, I, A, P, Q, or N or a derivative of any of them, still more preferably V, T, I, A, or N, or a derivative of any of them.
X3 is preferably G, W, S, A, L, F, T, R, N, Y, I, V, H, Q, K, D, or E, or a derivative of any of them, more preferably G, W, S, A, L, F, T, R, N, or Y, or a derivative of any of them, still more preferably G, W, or S, or a derivative of any of them.
X4 is preferably G, N, Y, I, R, E, F, S, W, H, D, T, P, A, V, L, Q, or K, or a derivative of any of them. X5 is preferably G, N, Y, I, R, E, F, S, W, H, D, T, P, A, V, L, Q, K, or C or a derivative of any of them.
For example, when KgpF is used as a prenylation enzyme, the prenylation enzyme can prenylate a peptide having the motif shown specifically below. In the following motifs, y means ClAc-D-Tyr.
VWG
VWW
VWS
VWI
VWC
VWT
VWR
VWQ
VWN
VWF
VWK
VWD
VWH
TWG
TWW
TWS
TWT
TWP
TWL
TWK
TWD
TWN
TWH
IWG
IWW
IWS
IWR
IWI
IWP
IWT
IWN
IWQ
IWE
IWH
AWG
AWW
AWS
AWH
AWV
AWD
AWL
AWT
AWR
AWQ
AWI
PWG
PWW
PWS
PWP
PWW
PWR
PWA
PWL
PWN
PWY
PWI
PWK
QWS
QWA
NWG
NWW
NWA
NWL
NWF
NWT
NWR
NWN
NWY
NWI
NWV
NWH
NWQ
NWK
NWD
NEW
NWP
NWS
SWS
FWF
FWS
HWS
HWG
HWD
WWQ
WWS
LWV
LWS
LWC
GWS
GWT
DWF
DWS
RWR
RWC
YWS
EWS
EWP
KWS
For example, when KgpF is used as a prenylation enzyme, the prenylation enzyme can also prenylate a peptide containing the motif shown below more specifically. In the following motif, y means ClAc-D-Tyr.
For example, when OltF is used as a prenylation enzyme, the prenylation enzyme can also prenylate a peptide containing the motif shown below specifically. In the following motif, y means ClAc-D-Tyr.
NWS
IWR
PWV
VWI
SWN
NWR
yWY
AWD
VWN
RWG
FWF
GWG
YWY
NWN
VWV
FWL
LWV
DWF
Further, for example when OltF is used as a prenylation enzyme, the prenylation enzyme can also prenylate a peptide containing the motif shown below specifically.
More specifically, at least peptides having the following amino acid sequence, as well as peptides shown later in Examples, can be prenylated.
This can be understood from the finding that for example KgpF prenylates two Trp residues contained in a main-chain cyclized peptide (Cyclic [WLNGDNNWSTP]) formed from a precursor peptide (KgpE) of Kawaguchipeptin A and composed of 11 residues to form a natural product Kawaguchipeptin A (refer to Tetrahedron, 1996, 52, 9025. ACIE, 2016, 55, 3596. Org. Biomol. Chem., 2016, 14, 9639.). The prenylation enzyme can also prenylate Fmoc-Trp-OH though at low efficiency. In the above description, Cyclic[ ] and c[ ] means that it is an amino acid sequence of a cyclic peptide and a left-end amino acid residue and a right-end amino acid residue in the bracket bond to each other.
Although the concentration of the prenylation enzyme is not particularly limited because it differs depending on the expression and purification conditions of the enzyme, it is, for example, from 0.1 to 1000 μM. The concentration of the prenylation enzyme can be made smaller by adjusting the expression and purification conditions of the enzyme.
(Peptide Library Having Phenotype Displayed on Genotype)
A display method is a system capable of linking a phenotype to a genotype encoding the sequence thereof through non-covalent bonding or covalent bonding to display the phenotype on the genotype and performing enrichment and amplification (selection) of active species by using a replication system reconstructed in a test tube.
Examples of it include phage display with Escherichia coli as a replication medium and yeast display.
In vitro display without using a prokaryotic or eukaryotic organism as a medium can also be used and the in vitro display permits more diverse library search than phage display. Examples of the in vitro display include ribosome display, cDNA display, and mRNA display.
(Method of Producing Peptide-Genotype Library)
One aspect of the present invention is a method of producing a peptide-genotype complex library including a step of preparing a peptide-genotype library including genotype-peptide complexe(s) and a step of bringing the peptide-genotype library into contact with a prenylation enzyme and prenylating at least one Trp or derivative thereof.
(Method of Producing Peptide-Genotype Library)
As the display method of the present invention, any method can be used, and mRNA display can be used preferably.
Described specifically, the method of producing a peptide-genotype complex library is preferably a method of producing a peptide-mRNA complex library. The step of preparing a peptide-genotype library includes a step of preparing a peptide-mRNA library by the mRNA display method; and the step of preparing a peptide-mRNA library includes a step of binding puromycin to the 3′ end of each RNA of the mRNA library to produce a puromycin-bound mRNA library and a step of translating the puromycin-bound mRNA library in a cell-free translation system and preparing a peptide-mRNA library including peptides having an amino acid sequence containing at least one Trp or derivative thereof.
The step of producing a puromycin-bound mRNA library is performed, more specifically, by binding puromycin to the downstream region of ORF (Open reading frame) of each mRNA when a mRNA library is prepared in the method of producing a peptide library described above. Puromycin may be bound to mRNA via a linker composed of a peptide or nucleic acid. An mRNA-peptide complex is formed by binding puromycin to the downstream region of ORF of mRNA and incorporating puromycin in a ribosome which has translated ORF of mRNA. Such a peptide-mRNA complex allows association between genotype and phenotype so that it can be applied to in vitro display.
(Peptide Library)
The present invention embraces a peptide library produced by the above-described production method. More specifically, the peptide library of the present invention includes an aspect of a peptide library and an aspect of a peptide-mRNA complex library including the above-described cyclic peptides.
(Screening Method)
One aspect of the present invention is a screening method for identifying a peptide that binds to a target material, including a step of bringing a peptide library and/or peptide-genotype complex library, preferably a peptide-mRNA complex library, each produced by the above-described method, into contact with a target material and a step of selecting a peptide that binds to the target material. The contact step in the screening method of the present invention may be performed by bringing a peptide library and/or peptide-genotype complex library, preferably a peptide-mRNA complex library, each produced by the above-described method into contact with a target material, followed by incubation.
The target material is not particularly limited in the present specification and examples include low molecular weight compounds, high molecular weight compounds, nucleic acids, peptides, proteins, sugars, lipids and the like. In particular, the library of the present invention can also be applied to the case where a target material has protease activity or is an intracellular molecule.
The target material immobilized onto, for example, a solid-phase support may be brought into contact with the peptide library and/or peptide-mRNA complex library according to the present invention. The term “solid-phase support” as used herein is not particularly limited insofar as it can immobilize the target material onto itself. Examples include microtiter plates, substrates, and beads made of glass, a metal, a resin or the like, nitrocellulose membranes, nylon membranes, and PVDF membranes. The target material can become immobilized onto such a solid-phase support in a known manner.
The target material and the library are brought into contact with each other in a buffer selected as needed and they are interacted while controlling pH, temperature, time and the like.
The screening method of the present invention includes a step of selecting a peptide that has bound to the target material. The peptide that has bound to the target material can be selected, for example, by labeling peptides detectably by a known method and after the contact step, washing the surface of the solid-phase support with a buffer, and then detecting the compound that has bound to the target material.
Examples of a detectable label include enzymes such as peroxidase, alkaline phosphatase and the like, radioactive substances such as 25I, 131I, 35S, 3H and the like, fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethyl rhodamine isothiocyanate, near infrared fluorescent materials and the like, light-emitting substances such as luciferase, luciferin, aequorin and the like, and nanoparticles such as gold colloid, quantum dot and the like. When an enzyme is used as the label, detection can also be achieved by bringing the substrate of the enzyme into contact with the enzyme to cause color development. It is also possible to detect by binding biotin to the peptide and then binding avidin or streptavidin labeled with an enzyme or the like.
The screening method can not only detect or analyze the presence/absence or degree of binding but also analyze the enhanced or inhibited activity of the target material and thereby identify a peptide having such enhancing or inhibiting activity. Such a method also allows identification of a peptide having physiological activity and useful as a drug.
In the screening method of the present invention, a display method can be applied to the step of selecting a peptide that binds to a target material and usable examples of such a display method include phage display using Escherichia coli as a replication medium, yeast display, and ribosome display, cDNA display and mRNA display, which are each an in vitro display not using a prokaryotic or eukaryotic organism as a medium. Particularly when the peptide-mRNA complex library is used in the screening method of the present invention, the mRNA display method can be applied to the step of selecting a peptide that binds to the target material. The term “mRNA display method” as used herein means a method of, in the translation of mRNA, binding the mRNA to a synthesized peptide to allow association between a phenotype (peptide) and a nucleic acid sequence (mRNA).
In the display method, a peptide-genotype complex library is brought into contact with a target molecule and after incubation, a peptide-genotype complex group bound to the target molecule is selected. This step can be carried out, similar to that used for the above-described selection of a peptide bound to a target material, for example by fixing a target material onto the surface of a solid phase and selecting a peptide-genotype complex trapped on the surface of the solid phase.
When as the display method, the mRNA display method is used, a group of cDNAs can be obtained by subjecting a group of peptide-mRNA complexes to a reverse transcription reaction. This cDNAs encode cyclic peptides binding to a target molecule.
By amplifying the cDNA group and transcribing the resulting group, an mRNA library can be obtained again. In the mRNA library thus obtained again, the concentration of a molecule that binds to the target molecule becomes higher than that of the first mRNA library. The molecule that binds to the target molecule can therefore be enriched gradually by repeating the above step in a plurality of times.
The amino acid sequence of the enriched cyclic peptide can be determined by analyzing the sequence of the cDNA so that a cyclic peptide having high affinity for the target molecule can be produced based on the sequence information.
The RaPID system (Yamagishi, Y. et al., Chemistry & biology, 2011, 18 (12), 1562-70) is one example of a screening system having a FIT system, used in Examples described later, and mRNA display in combination. The RaPID system having a genetic code reprogramming technology using the FIT system and the mRNA display method in combination has enabled screening with a peptide library including nonproteins.
(Screening Kit)
The present invention also provides a peptide screening kit. One aspect of the screening kit of the present invention includes a peptide library and/or peptide-mRNA complex library produced by the production method of the present invention.
The screening kit of the present invention includes, in addition to the above-described peptide library and/or the peptide-mRNA complex library, a reagent and apparatus necessary for detecting binding of the peptide or peptide-mRNA complex and a target material. Examples of such a reagent and apparatus include, but not limited to, solid-phase supports, buffers, labeling reagents, enzymes, enzyme reaction terminator solutions, and microplate readers.
(Method of Producing Peptide)
The present invention also provides a method of producing a prenylated peptide, including a step of bringing a peptide having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating the at least one Trp residue or derivative residue thereof.
Although a method of obtaining the peptide having an amino acid sequence containing at least one Trp or derivative thereof is not particularly limited, examples include chemical synthesis, preferably a method of synthesizing using a solid-phase synthesis method. One of the preferred aspects of the method of producing a peptide according to the present invention includes a step of synthesizing, by chemical synthesis, a peptide having an amino acid sequence containing at least one Trp or derivative thereof and a step of bringing the peptide having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme and prenylating the at least one Trp residue or derivative residue thereof.
The method of producing a peptide according to the present invention can be carried out based on the above-described method of producing a peptide library according to the present invention. Described specifically, in the prenylation step, it is preferred to bring a peptide having an amino acid sequence containing at least one Trp or derivative thereof into contact with a prenylation enzyme in the presence of a donor for prenyl group in the presence or absence of a cofactor.
Although the donor for donating a prenyl group is not particularly limited, examples include dimethylallyl diphosphate (DMAPP), isoprenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and phytyl diphosphate (PDP). These donors for donating a prenyl group may be used either singly or in combination of two or more.
Although the cofactor is not particularly limited, examples include magnesium ion, copper ion, iron ion, manganese ion, molybdenum ion, nickel ion, selenium ion, zinc ion and the like. They may be used either singly or in combination of two or more. Of these, magnesium ion is preferred. Whether the cofactor is used or not in the method of producing a peptide according to the present invention may be selected as needed, depending on the prenylation enzyme to be used.
(Translation System)
In the present invention, the cell-free translation system may contain an elongator tRNA and may further contain an initiator tRNA. As described above, the present inventors have developed the translation system in which N1N2N3 encodes an arbitrary amino acid by codon reassignment making use of flexizyme. In a natural translation system, tRNAs having an anticodon corresponding to each amino acid are present and the tRNAs have intrinsic sequences respectively also in a region other than the anticodon loop.
When an arbitrary amino acid is reassigned to any of N1N2N3s by making use of flexizyme, all the tRNAs may be artificial. In this case, elongator tRNAs corresponding to respective N1N2N3s to be added to the translation system may have base sequences 80% or more, 85% or more, 88% or more, or 90% or more in full length identical to one another. This means that a group of elongator tRNAs almost equal in their sequences except for the sequences of anticodons may be used. The elongator tRNA group may have base sequences identical to one another except for anticodon loops. The elongator tRNAs corresponding to respective N1N2N3s to be added to the translation system may have sequences 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 98% or more, or 99% or more identical to one another except for anticodon loops.
The term “anticodon loop” as used herein means a loop portion of a single strand of a tRNA containing an anticodon. The sequence of the anticodon loop can be determined as needed by those skilled in the art so as to complement the codon-anticodon interaction.
The translation system thus obtained includes, as tRNAs, only one kind of initiator tRNAs and one kind of elongator tRNAs (that is, tRNAs having almost the same base sequence except for anticodons) so that the reactivity of the tRNAs becomes uniform and predetermined peptides can therefore be obtained with good reproducibility.
According to the translation system of the present invention, a library including 1×1012 to 1×1013 or more respectively different peptides can be produced based on the diversity of N1N2N3 in the mRNA.
The translation system of the present invention may be used for transcription after addition of a factor necessary for transcription.
The translation system of the present invention is suited for use in the method of producing a peptide library according to the present invention described above.
All the disclosures of the patent documents and reference documents cited herein are incorporated herein in their entirety by reference.
The present invention will hereinafter be described specifically based on Examples, but the present invention is not limited to or by them. Those skilled in the art can change the present invention into various aspects without departing from the gist of the present invention and such a change is also embraced within the scope of the present invention.
1. FIT System v7S/v8.0.3
The FIT system has the following composition:
50 mM HEPES-KOH (pH 7.6); 12 mM magnesium acetate; 100 mM potassium acetate; 2 mM sperm idine; 20 mM creatinine phosphate; 2 mM DTT; 2 mM ATP; 2 mM GTP; 1 mM CTP; 1 mM UTP; 0.1 mM 10-formyl-5,6,7,8-tetrahydrofolic acid; 0.5 mM 15 proteinogenic amino acids other than Met, Lys, Gln, Trp, and Glu; 1.5 mg/ml E. coli total tRNA; 0.73 μM AlaRS; 0.03 μM ArgRS; 0.38 μM AsnRS; 0.13 μM AspRS; 0.02 μM CysRS; 0.06 μM GlnRS; 0.23 μM GluRS; 0.02 μM GlyRS; 0.02 μM HisRS; 0.4 μM IleRS; 0.04 LeuRS; 0.11 μM LysRS; 0.03 μM MetRS; 0.68 μM PheRS; 0.16 μM ProRS; 0.04 μM SerRS; 0.09 μM ThrRS; 0.03 μM TrpRS; 0.02 μM TyrRS; 0.02 μM VaIRS; 0.6 μM MTF; 2.7 μM IF1; 0.4 μM IF2; 1.5 μM IF3; 0.26 μM EF-G; 10 μM EF-Tu; 10 μM EF-Ts; 0.25 μM RF2; 0.17 μM RF3; 0.5 μM RRF; 0.1 μM T7 RNA polymerase; 4 pg/ml creatine kinase; 3 pg/ml myokinase; 0.1 μM pyrophosphatase; 0.1 μM nucleotidediphosphatase kinase, and 1.2 μM ribosome.
2. Preparation of ClAc-D/L-Trp/Tyr-tRNAfMetCUA
In 83.3 mM HEPES-KOH (pH 8.0), 3 μL of 83.3 μM tRNAfmetCUA and 3 μL of 83.3 μM flexizyme were heated at 95° C. for 2 minutes and then cooled to room temperature in 5 minutes. To the resulting RNA solution was added 2 μL of 3 M MgCl2 and the resulting mixture was incubated on ice for 5 minutes. Then, 2 μL of 25 mM ClAc-D-Trp-CME, ClAc-L-Trp-CME, ClAc-D-Tyr-CME, or ClAc-L-Tyr-CME (in DMSO) was added to the reaction mixture, followed by incubation on ice for 2 hours. The reaction was terminated by adding 40 μL of 0.3 M sodium acetate (pH 5.2) and tRNA was collected by ethanol precipitation. Precipitate was washed twice with 70% ethanol and 0.1 M sodium acetate (pH 5.2) and once with 70% ethanol. After air drying for 10 minutes, the precipitate was dissolved in 1.0 μL of 1.0 mM sodium acetate and the resulting solution was used for the following translation reaction.
3. Prenylation Enzyme
With regards to each of KgpF and OltF, heterogeneous co-expression of a gene synthesized after optimizing the codon of a gene derived from a producing strain was performed in Escherichia coli, followed by purification to obtain a prenylation enzyme.
4. MALDI-TOF Analysis
The analysis was performed using UltrafleXtreme (Bruker Daltonics) or Autoflex II (Bruker Daltonics) and peptide calibration standard II (Bruker Daltonics).
In addition, reagents and the like disclosed in WO2014/181888 and WO2015/030014, particularly those disclosed in Examples thereof were used as needed also in the present Examples.
Three kinds of thioether-closed artificial cyclic amino acid sequences YG280 to 282 derived from the sequence of Kawaguchipeptin, a natural substrate, (which may also be called “natural substrate sequence”) were designed.
(In the formula, shown in the parentheses is an amino acid sequence).
For three kinds of thioether-closed artificial cyclic peptides (YG280 to 282), peptides whose N-terminal Trp as an initiator residue had a steric configuration of D-form and those of L-form were synthesized. In short, six kinds in total of peptides were obtained by translation and synthesis under (Conditions 1) of the artificial translation system developed by the present inventors. The peptides thus obtained were then reacted with 14.7 μM KgpF at 37° C. for 18 hours in the system shown in (Conditions 2). “DMAPP” used in (Conditions 2) is dimethylallyl pyrophosphoric acid.
(Conditions 1)
(Conditions 2)
The above reaction products were analyzed by MALDI-TOF-MS. The results were as shown below.
For all the three kinds of peptides tested, products having a molecular weight increased by that corresponding to prenylation were observed when they were treated with KgpF. This has suggested that the above peptides become a substrate of KgpF.
Although the peptides had a Trp at two positions therein, prenylation occurred at only one position.
Among the three kinds of peptides tested, the peptide having the YG280 sequence with a steric configuration of the N terminal Trp of D-form showed the most excellent modification efficiency by prenylation, so that the tests were continued further while focusing on the YG280 sequence.
Based on the results of Example 1, the prenylation reaction conditions were optimized by using the peptide having a YG280 sequence and gradually increasing the concentration of KgpF.
The YG280 sequences having the steric configuration of the N terminal Trp serving as an initiation residue of D-form and those of L-form were synthesized, so that two kinds of peptides were prepared in total. These two kinds of artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 3) developed by the present inventors. The peptides thus obtained were then reacted with 53, 160, and 580 μM KgpF in a system shown in (Conditions 4) at 37° C. for 18 hours.
(Conditions 3)
(Conditions 4)
In the system using 160 μM KgpF, slight precipitation was found, while in the system using 580 μM KgpF, much precipitation was found.
The above reaction products were analyzed by MALDI-TOF-MS. The results are as shown below.
With an increase in the concentration of KgpF, a peak showing the modification product became more intense correspondingly.
The peptides having an N-terminal Trp in D-form as the steric configuration were superior in prenylation modification efficiency to those having that in L-form.
When the peptides having an N-terminal Trp in D-form as the steric configuration were reacted with 580 μM KgpF, almost all the peptides underwent prenylation modification (there were only few peaks showing non-prenylated peptides).
The peptides had a Trp at two positions but prenylation occurred only at one position.
It has been found that using a highly-concentrated KgpF enables efficient prenylation of a thioether-closed artificial cyclic peptide.
In Examples 1 and 2, prenylation occurred only at one position, though the peptide had a Trp at 2 positions. To determine the position where prenylation occurred, the peptides (YG298, 280) whose Trp residues in the YG280 sequence had each been mutated into Tyr and the peptide (YG290) having no Trp residue in a random region were designed and presence or absence of progression of modification with KgpF was assessed.
The above three kinds of artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 5) developed by the present inventors. The peptides thus obtained were then reacted with KgpF in the system shown below in (Conditions 6) at 37° C. for 18 hours.
(Conditions 5)
(Conditions 6)
The above reaction products were analyzed by MALDI-TOF-MS. As a result, it has been confirmed that no modified product was found even after the peptide YG298 having the N-terminal Trp residue remained therein or the peptide YG290-CM11 having no Trp residue in the peptide chain thereof was reacted with 580 μM KgpF, while the peptide ClAc-D-Tyr-initiated YG280 having the Trp residue remained in the chain (8th residue) was modified at an efficiency equal to that of the peptide of the original sequence (YG280). This has suggested that the Trp residue in the peptide chain of the peptide YG280 underwent prenylation modification.
The YG280 sequence derived from a natural substrate sequence was a thioether-closed cyclic peptide composed of 12 residues. It was therefore studied whether the thioether-closed cyclic peptides (YG310 and 311) having a shorter sequence and the thioether-closed cyclic peptides (YG312 to 314) having a longer sequence than that of YG280-1 used as a standard could be modified with KgpF or not.
The above six kinds of artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 7) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 8) at 37° C. for 18 hours.
(Conditions 7)
(Conditions 8)
The above reaction products were analyzed by MALDI-TOF-MS. As a result, it has been found that although there was an increase or decrease in the modification efficiency, depending on the ring size, all the sequences tested were modified with KgpF. This has proven that KgpF can modify thioether-closed artificial cyclic peptides having various ring sizes.
In the natural substrate sequence and YG280, amino acid residues present upstream and downstream of Trp to be modified were Asn and Ser, respectively. The three kinds of amino acid sequences (YG300, 304, and 305) were designed by changing the flanking sequence of Trp and whether they could be modified with KgpF or not was studied.
The above three kinds of artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 9) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 10) at 37° C. for 18 hours.
(Conditions 9)
(Conditions 10)
The reaction products were analyzed by MALDI-TOF-MS. As a result, it was found that the three kinds of the artificial peptides tested were each modified by prenylation.
In addition, the following cyclic peptides (YG288-S2iL8, YG289-S2iD7, YG292-PaktiL5, and YG297-aMD4) were also obtained similarly by translation and synthesis and reacted with DMAPP in the presence of KgpF. It was observed by MALDI-TOF-MS that they were modified with a prenyl group.
To closely investigate the influence of the flanking sequence of Trp on the modification efficiency with KgpF, 13 kinds of sequences having, instead of the flanking sequence of W, the NWS or NNWST sequence found in Kawaguchipeptin, a natural substrate, were designed (Table 5) and whether they could be modified with KgpF or not was studied.
The above artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 11) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 12) at 37° C. for 18 hours.
(Conditions 11)
(Conditions 12)
The above reaction products were analyzed by MALDI-TOF-MS. As a result, it has been found that a modification efficiency with KgpF showed a marked improvement by replacing the flanking sequence of W with the NWS or NNWST sequence found in the natural substrate.
What kind of a flanking sequence of W would make the resulting peptide excellent and preferable in prenylation with KgpF was studied further comprehensively.
Here, the following mutants were prepared comprehensively by replacing the flanking sequence of W of peptide YG326 with another amino acid residue and whether they could be prenylated with KgpF or not was studied.
The peptides in which N (X in Mutant I of YG326) upstream of W contained in the sequence of peptide YG326 and S (X in Mutant II of YG326) downstream of the W were replaced with various amino acid residues shown along the abscissa of the graphs shown in
The graphs shown in
The level of the modification efficiency of N which is a natural upstream sequence is indicated by a dotted line.
The following respective compounds having D, E, K and Y as the amino acid upstream of W and the following compound having P as the amino acid downstream of W were also synthesized as in Example 6 and a prenylation reaction was performed as in Example 6.
Observation by MALDI-TOF-MS showed that the above compounds YG280-DWS, YG280-EWS, YG280-KWS, YG280-YWS, and YG280-NWP were modified with a propyl group.
It has been found in general that amino acid residues having a variety of properties such as acidity, basicity, affinity, hydrophobicity and the like were tolerated both at the upstream position and the downstream position.
Based on the results obtained in Example 7, it becomes possible to design some flanking sequences giving a higher modification efficiency than the natural NWS flanking sequence. Here, the 10 kinds of flanking sequences were newly designed (the abscissa of
The above reaction products thus obtained were analyzed by MALDI-TOF-MS. As a result, it has been found that almost all the peptides thus analyzed showed a modification efficiency higher than that of the peptide having a natural NWS sequence (
Based on the results of the modification efficiency of the flanking sequence obtained in Example 7, a peptide whose amino acid residue upstream of W was fixed to the most preferable Val and amino acid sequence on the downstream side was changed was designed. Designed were mutants obtained by replacing the X portion of VWX with the 6 kinds of amino acids (G, W, S, F, N, and K). The resulting peptides were synthesized as in Example 6 and a prenylation reaction was performed as in Example 6.
The above reaction products thus obtained were analyzed by MALDI-TOF-MS. As a result, it has been found that they showed a modification efficiency equal or greater than the natural NWS (
The artificial cyclic peptides Clone-M1 to M9 shown below were obtained by translation and synthesis in the artificial translation system (Conditions 13) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 14) at 0° C. for 20 hours.
(In the above formulas, “y” represents D-Tyr).
(Conditions 13)
After the above reaction was performed, the reaction product was incubated at 25° C. for 12 minutes.
Then, a cyclization reaction was performed under the following conditions.
(Conditions 14)
Shown in Table 6 are 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.
Reaction of the artificial cyclic peptide shown in Table 7 was made under conditions similar to (Conditions 11) and (Conditions 12) except that v8s/v11.1 was used as FIT system, KgpF v 3.1 was used as a prenylation modification enzyme, and the concentration of the KgpF v 3.1 was set at 10 μM. Here, KgpF v 3.1 is an enzyme having a higher purification degree and more improved specific activity than KgpF used under (Conditions 12).
Shown in Table 7 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.
YLNGDNNWSTPC
YLNGDNNWSPC
YSNFRIWRYSNSSC
YHDYRIWRYHTYPC
YWILITWPLVRRKC
YIRRPWVPIIYLGC
YTFRDVWIFYGSLLSRC
YISWNEFNSPNWRFITC
YWYYAWDQTYKAFPC
YRQFNRRTHEVWNLDC
The artificial cyclic peptides shown in Table 8 and Table 9 were obtained by translation and synthesis in the artificial translation system (Conditions 15) developed by the present inventors. The peptides thus obtained were then reacted with 60 μM KgpF in the system shown in (Conditions 16) at 37° C. for 18 hours.
(Conditions 15)
(Conditions 16)
Shown in Table 8 and Table 9 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.
CYPR
CYP
PCY
YPC
YPC
YHPC
YHPC
YHTPC
YHTPC
YHTYPC
YHTYPC
YHTYQPC
YHTYQPC
YHTYQNPC
YHTYQNPC
The artificial cyclic peptides shown in Table 10 were obtained by translation and synthesis in the artificial translation system (Conditions 17) developed by the present inventors. The peptides thus obtained were then reacted with 60 μM KgpF in the system shown in (Conditions 18) at 37° C. for 18 hours.
(Conditions 17)
(Conditions 18)
Shown in Table 10 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.
HDYRVYHTYPC
HDYRYHTYPC
DYRYHTYPC
YRYHTYPC
RYHTYPC
HTYPC
TYPC
YPC
PC
C
YHTYPV
C
The artificial cyclic peptides shown below were obtained by translation and synthesis in the artificial translation system (Conditions 19) developed by the present inventors. The peptides thus obtained were then reacted with 160 μM KgpF in the system shown in (Conditions 20) at 0° C. for 20 hours.
The reaction products were analyzed by MALDI-TOF-MS. As a result, it has been confirmed that the artificial cyclic peptides shown below were modified by prenylation. As described above, the fact that prenylation proceeded in peptides having various sequences means that a peptide library including peptides having higher cell membrane permeability can be obtained efficiently.
(Conditions 19)
After the above reaction was performed, the reaction product was incubated at 25° C. for 12 minutes.
Then, a cyclization reaction was performed under the following conditions.
(Conditions 20)
(In the above formulas, “y” represents D-Tyr).
(In the above formulas, “y” represents D-Tyr).
(In the above formulas, “y” represents D-Tyr).
(In the above formulas, “y” represents D-Tyr).
(In the above formulas, “y” represents D-Tyr).
In order to prove that not only KgpF but also another enzyme having homology with it could modify a thioether-closed artificial cyclic peptide, it was studied whether or not a thioether-closed artificial cyclic peptide could be modified with an OltF enzyme.
The 6 kinds of thioether-closed artificial cyclic amino acid sequences (YG413 to 417 and 419) derived from the sequence of OscE1 and OscE2 presumed to be natural substrates of OltF were designed.
The above artificial cyclic peptides were obtained by translation and synthesis in the artificial translation system (Conditions 21) developed by the present inventors. The peptides thus obtained were then reacted with 24 or 72 μM OltF in the system shown in (Conditions 22) at 37° C. for 18 hours.
(Conditions 21)
(Conditions 22)
The above reaction products thus obtained were analyzed by MALDI-TOF-MS. As a result, a product modified by prenylation was observed in any of the peptides. It has been found from the above fact that OltF, similar to KgpF, has an ability of modifying not only a natural main-chain cyclized peptide but also a thioether-closed artificial cyclic peptide. This has suggested that a group of enzymes having high homology with OltF or KgpF can modify a thioether-closed artificial cyclic peptide similarly to them and therefore can be used as the prenylation enzyme of the present invention.
The artificial cyclic peptides shown in Table 11 and Table 12 were obtained by translation and synthesis in the artificial translation system (Conditions 23) developed by the present inventors. The peptides thus obtained were then reacted with 9.13 μM OltF in the system shown in (Conditions 24) at 37° C. for 18 hours. It is to be noted that OltF v 2.1 is an enzyme prepared from OltF used in (Conditions 22) while changing a purification manner.
(Conditions 23)
(Conditions 24)
Shown in Table 11 and Table 12 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.
YLNGDNNWSPC
YLNGDNNWSTC
YSNFRIWRYSNSSC
YHDYRIWRYHTYPC
YIRRPWVPIIYLGC
YTFRDVWIFYGSLLSRC
YISWNEFNSPNWRFITC
YWYYAWDQTYKAFPC
YRQFNRRTHEVWNLDC
YCDVSGRWGYFPC
Sequence The number of prenyl groups added
YNNRERGSDFFWFFPC
YNNRERGSDGGWGGPC
YNNRERGSDYYWYYPC
YNNRERGSDNNWNNPC
YNNRERGSDVVWVVPC
YISWNEFNSYFWLPITC
YNNRERGSDYFWLPPC
YRQFNRRTHYFWLPDC
(Design of mRNA Library)
As shown in Table 13, an mRNA library was designed.
In Table 13, from top to bottom the sequence identifiers are SEQ ID NOs: 503 to 512.
In the mRNAs in Table 13, amino acids were reassigned based on the genetic code table shown below in Table 14. Here, Trp is reassigned to UGG of the mRNA in Table 13. XXX was RYU and CMG (RYU and CMG were present at 2:1 (RYU: CMG)) or RYU. It is to be noted that in RYU, Rs each independently represent G or A and Ys each independently represent U or C; in CMG, Ms each independently represent A or C; and in NNK, Ks each independently represent G or U, and N is an arbitrary base.
(Preparation of mRNA Library)
Extension was achieved as shown in Table 15 by keeping at 95° C. for 1 minute, keeping at 61° C. for 1 minute and then keeping at 72° C. for 1 minute. Keeping at 61° C. for 1 minute and then keeping at 72° C. for 1 minute were performed with 5 cycles.
It is to be noted that the sequence of the primer T7YGM.F46 v1tr.F46 is as follows:
<Composition of 10 mL Scale System>
Taq Polymerase
PCR (polymerase chain reaction) was performed, as shown in Table 16, by keeping at 95° C. for 40 seconds, keeping at 65° C. for 40 seconds, and then keeping at 72° C. for 40 seconds. Keeping at 95° C. for 40 seconds, keeping at 65° C. for 40 seconds and then keeping at 72° C. for 40 seconds were performed with 4 cycles.
It is to be noted that the following are the respective sequences of the primers T7YGM.F46 v1tr.F46 and rCNSGGVSan13.R39.
As shown in
(Purification of mRNA Library)
Preparation of an mRNA library was successfully achieved by performing in vitro transcription (4 mL scale) of the resulting PCR product with T7 RNA polymerase and purifying the products by electrophoresis under conditions of 12% denaturing-PAGE, 230V, and 70 minutes. As shown in
To prove that prenylating modification could be performed not only in a liquid phase but also in a solid phase, it was studied whether or not an artificial cyclic peptide could be modified with a KgpF enzyme immobilized onto the surface of magnetic beads via a tag.
(Immobilization of KgpF onto Magnetic Beads)
Dynabeads His-Tag Isolation and Pulldown (Invitrogen) was brought into contact with KgpF having a His-tag added N terminal and the KgpF was immobilized onto the magnetic beads. As shown in
(Modification of Artificial Cyclic Peptide with KgpF Immobilized onto Magnetic Beads)
The artificial cyclic peptides shown in Table were synthesized under (Conditions 25) and were brought into contact with KgpF in solid phase under (Conditions 26). In addition, the same peptides were reacted in liquid phase under conditions shown in (Conditions 13) and (Conditions 14) except that the prenylation reaction time was changed to 14 hours.
(Conditions 25)
After the above reaction was performed, the reaction product was incubated at 25° C. for 12 minutes.
Then, a cyclization reaction was performed under the following conditions.
(Conditions 26)
The solution obtained by the reaction was analyzed by MALDI-TOF-MS. As shown in the table, although the KgpF concentration is lower in solid phase, the modification efficiency is 100%, similarly to that in liquid phase (no peak of start material was found). This has revealed that modification can be achieved in solid phase similarly to that in liquid phase.
Shown in Table 17 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.
A library was prepared based on the design of the mRNA library shown in Example 17 in a manner similar to that of Example 17 except that the XXX portion was replaced with RYU.
A peptide-mRNA complex library was constructed from the above mRNA library according to the scheme shown in
Then, the compound library was provided for a step of screening cyclic peptides having interaction with an enzyme MetAP1 (Human) as in the scheme shown in
The above results have revealed that a compound library can be provided by the production method of the present invention using a prenylation enzyme.
Shown in Table 18 and Table 19 are: 5: strong intensity, 4: medium intensity, 3: weak intensity, 2: very weak intensity, 1: no peak, and N/A: not observed.
With regards to the prenylated cyclic peptides identified as a result of screening with MetAP1 as a target, their binding ability to the target was assessed by qualitative pulldown assay. After synthesis of Met3, Met9, Met11, Met12, Met13 and Met14 while displaying each of the sequences on mRNA, the Trp residue in the sequence was prenylated. The prenylated peptide-mRNA complex thus obtained was pulled down with magnetic beads and then, a recovered mRNA amount was determined by RT-qPCR. The results are shown in
In the bar graph of
(Synthesis of Prenylated Cyclic Peptide by Using Fmoc Solid-Phase Synthesis and Prenylation with KgpF)
After chemical synthesis of respective cyclic peptides having sequences Met2 (SEQ ID NO: 361), Met3-1 (SEQ ID NO: 360), Met3a-L (SEQ ID NO: 362), Met11 (SEQ ID NO: 363) and Met13 (SEQ ID NO: 364) by Fmoc solid-phase synthesis, they were treated with KgpF in a test tube to prepare prenylated cyclic peptides. The prenylation was performed under the following Conditions 27.
(Conditions 27)
As shown in
(MetAP1 Inhibition Test of Prenylated Cyclic Peptide)
A MetAP1 inhibition test was performed in vitro using the prenylated Met2, Met3a-L, and Met3-1 at concentrations of 1 μM, 5 μM or 10 μM in the presence of a MetAP1 cofactor Mn2+ or Co2+. In the test, enzymatic activity was measured by detecting probe fluorescence, that is, fluorescence emitted depending on the enzymatic activity of MetAP1. The measurement results of fluorescence intensity are shown in
(Conditions)
After the reaction, the fluorescence intensity of the probe was measured using a plate reader.
Prenylation reaction was performed using the cyclic peptide shown below under conditions shown in (Conditions 27) in the presence or absence of Mg2+ at a concentration of EDTA adjusted to 0 mM, 1 mM or 10 mM. The cyclic peptides shown below were obtained by the synthesis using solid-phase synthesis.
(Conditions 27)
The reaction products were analyzed by MALDI-TOF-MS. As a result, it has been found that the reaction proceeded dependently on Mg2+ when KgpF was used as a prenylation enzyme (refer to
A prenylation test with KgpF was performed using a cyclic peptide containing an amino acid in D-form as a non-proteinogenic amino acid. The cyclic peptide containing an amino acid in D-form (X in the following formula) is represented by the following formula.
The above artificial cyclic peptides containing an amino acid in D-form were obtained by translation and synthesis under the following (Conditions 28) in the artificial translation system developed by the present inventors. The translated mixtures thus obtained were then reacted (prenylated) under the following (Conditions 29).
(Conditions 28)
(Conditions 29)
The reaction products were analyzed by MALDI-TOF-MS. As a result, it has been confirmed that the respective artificial cyclic peptides containing, as X, Ile in D-form, Phe in D-form, Trp in D-form and Tyr in D-form were modified by prenylation (refer to
By using a cyclic peptide containing a non-proteinogenic amino acid Trp, a prenylation test of the Trp with OltF or KgpF was performed. The cyclic peptide used is as follows. In the formula, X is Trp in D-form.
The above artificial cyclic peptide containing Trp in D-form was obtained by translation and synthesis under the following (Conditions 30) in the artificial translation system developed by the present inventors. The translated mixture thus obtained was then reacted (prenylated) under the following (Conditions 31) at 37° C. for 20 hours.
(Conditions 30)
(Conditions 31)
The reaction product was analyzed by MALDI-TOF-MS. As a result, it has been confirmed that the artificial cyclic peptide containing, as X, Trp in D-form was modified by prenylation (refer to
The cyclic peptide shown below was obtained by translation and synthesis under the following (Conditions 32) in the artificial translation system developed by the present inventors. The translated mixture thus obtained was then reacted (prenylated) under the following (Conditions 33).
(Conditions 32)
(Conditions 33)
The reaction product was analyzed by MALDI-TOF-MS. As a result, it has been found that a Trp at two positions, that is, at the N-terminal position and in the sequence could be prenylated by using OltF as a prenylation enzyme (refer to
In a manner similar to that described above, prenylation of AY015 (SEQ ID NO: 372), AY016 (SEQ ID NO: 373), AY017 (SEQ ID NO: 374), AY018 (SEQ ID NO: 375), AY019 (SEQ ID NO: 376) and AY020 (SEQ ID NO: 377) (refer to
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-196102 | Oct 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/040975 | 10/17/2019 | WO |
