JP6229966B2 - MATE activity inhibitory peptide - Google Patents

Description

  The present invention relates to a peptide or the like that inhibits the multidrug efflux transport activity of the multidrug efflux transporter MATE protein.

  In recent years, multidrug excretion transporters that widely recognize drugs with different structures and actions and excrete them from cells have attracted attention. Multidrug efflux transporters exist widely from prokaryotes to higher eukaryotes and are involved in various drug resistances. For example, a plurality of antibiotics and the like are actively discharged from pathogenic bacteria, and multidrug resistance is given to the pathogenic bacteria. In addition, the anticancer drug may be discharged from the cancer cells to suppress the effect of the anticancer drug.

As a multidrug efflux transporter, MATE (Multidrug And Toxic Compound Extrusion) protein is known (Non-patent Documents 1 and 2). It has approximately 450 amino acid residues and 12 transmembrane helices, and transports drugs and sodium ions and protons in opposite directions.
Bacterial MATE can excrete a wide range of antibiotics and contribute to drug resistance. Based on previous biochemical studies in bacteria, MATE recognizes cationic organic compounds, especially pigment compounds containing quaternary ammonium in the aromatic ring (eg, ethidium bromide, acriflavine hydrochloride). Has been suggested. In addition, fluoroquinolone antibiotics and aminoglycoside antibiotics (for example, kanamycin and streptomycin) are also recognized by MATE as substrates (Non-patent Document 3).

MATE is presumed to be responsible for drug excretion especially in Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae. If an inhibitor of MATE can be obtained, the effects of drugs targeting Gram-positive bacteria can be reduced. It is considered effective to promote.
In addition, homologues have been found in humans and plants in MATE.

Miyamae, S. et al., Antimicrobial agents and chemotherapy 45, 3341-3346 (2001) Bachoual, R. et al., Antimicrobial agents and chemotherapy 44, 1842-1845 (2000) Kuroda, T. and Tsuchiya, T. Biochimica et biophysica acta 1794, 763-768, doi: 10.1016 / j.bbapap.2008.11.012 (2009)

  An object of this invention is to provide the peptide which inhibits the multidrug efflux transport activity of MATE protein.

As a result of repeated studies to solve the above problems, the present inventors have found that a specific peptide consisting of a cyclic portion having about 3 to 15 amino acids and a tail portion having 1 or more amino acids is a multidrug excretion of MATE. It discovered that activity was inhibited and came to complete this invention.
That is, the present invention
[1] A peptide that inhibits the multidrug efflux transport activity of MATE protein, which has a cyclic part consisting of 3 to 15 amino acids and a tail part of one or more amino acids;
[2] The peptide according to [1], wherein the cyclic moiety has at least one of the following characteristics (i) to (iii):
(i) the N-terminus is F, Y, W, H, or an enantiomer thereof, and is bonded to C at the C-terminus to form a ring;
(ii) at least two amino acids are hydrophobic amino acids; and
(iii) contains 1 or 2 Y;
[3] The peptide according to the above [1], wherein the cyclic moiety is composed of 7 amino acids and has at least one of the following characteristics (a) to (f):
(a) the N-terminus is F, Y, W, H, or an enantiomer thereof, and is bonded to C at the C-terminus to form a ring;
(b) the second from the N-terminus is V, L, or T;
(c) the third from the N-terminus is F, Y, or H;
(d) the fourth from the N-terminus is S, A, P, N, or Q;
(e) the fifth from the N-terminus is A, V, S, L, or T; and
(f) the sixth from the N-terminus is A, V, F, or Y;
[4] The peptide according to [1] above, wherein the cyclic part includes a sequence with VYSAV, or a sequence in which one amino acid is deleted, added, or substituted in this sequence;
[5] The above-mentioned [4], wherein the sequence in which one amino acid is deleted, added, or substituted in the VYSAV sequence has at least one of the features (b) to (f) described in [3] above The peptide according to 1.
[6] The peptide according to any one of [1] to [5] above, wherein the tail portion includes 7 or more amino acids and has at least one of the following characteristics (I) and (II):
(I) 30% or more of the amino acids are hydrophobic amino acids; and
(II) including a site where two or more amino acids are consecutive hydrophobic amino acids;
[7] Any of the following peptides:
FVYSAVCLYVGSLYPCG;
FLYNAYCLWLAYCVNSCG;
FTHPIFCYPSADLCG;
FTYSAFCYAIANIAYCG;
fQWQCHIFTNLALTCG;
fVYSAVCLYVGSLYSCG;
fVYSAVCLYVGSLYSG;
fLYNAYCLWLAYCVNSCG;
fVYSAVCYSIAAAAAAARTG;
fVYSAVCYSIAAAAAAA;
fVDASACSFVNLWLTCG;
fSVACSAFVRIAHHASCG;
fTTYSAFCYAIANIAYCG; and
fTYSAFCYAIANIAYCG
(In the formula, F at the N-terminal represents L-phenylalanine and f represents D-phenylalanine. F or f is N-chloroacetylated, and the chloroacetyl group is bonded to C at the most N-terminal side to form a ring. May be formed);
[8] a nucleic acid encoding the peptide according to any one of [1] to [7] above; and [9] an anti-peptide comprising the peptide according to any one of [1] to [7] above. The present invention relates to a therapeutic effect enhancer for cancer drugs or drugs for treating multidrug-resistant bacterial infections.

  According to the peptide of the present invention, the drug excretion / transport activity by MATE can be suppressed. Therefore, by administering together with a multidrug-resistant bacterial infection therapeutic agent or an anticancer agent, there is a possibility that these drugs can be prevented from being discharged outside the cells, and the therapeutic effect of the drugs can be enhanced.

FIG. 1 shows the reprogramming and selection scheme of the genetic code. FIG. 1A shows the reprogrammed genetic code used for this selection used to replace formylmethionine in the starting CAU codon box with N-chloroacetyl-L-phenylalanine or N-chloroacetyl-D-phenylalanine. . White boxes represent codons that do not appear in the NNK library region. FIG. 1B is a conceptual diagram showing non-standard peptide production starting with N-chloroacetyl-L-phenylalanine or N-chloroacetyl-D-phenylalanine. In the peptide thus obtained, a macrocyclization reaction with a downstream cysteine occurs spontaneously. FIG. 1C is a conceptual diagram illustrating one of the rounds in the selection of cyclic peptides using affinity for the PfMATE protein. FIG. 2 shows the progression of selection and clonal assays performed at 4 ° C. and 37 ° C. FIG. 2A shows the enrichment of L- and D-pools binding to PhMATE in a 4 ° C. selection. FIG. 2B shows the enrichment of L- and D-pools binding to PhMATE in a 37 ° C. selection. FIG. 2C shows the binding of individual peptide-mRNAs to immobilized PfMATE. FIG. 3 shows the structure and MALDI spectrum of a chemically synthesized peptide. FIG. 4 shows the inhibitory activity of selected peptides. The graph shows the effect of various doses of peptides D1, D5, D8 and L6 on the ability of cells to excrete ethidium bromide. FIG. 5a shows the chemical formulas of the peptides MaD5 and Mad3S of the present invention. FIG. 5b shows a diagram in which MaD5 and MaD3S are co-crystallized with PfMATE, a complex X-ray analysis structure is performed, and visualized with a space filling model. FIG. 5 c shows the results of examining the inhibitory activity of each peptide on the ethidium bromide excretion ability of cells. FIG. 6 shows measured values when PhMATE inhibits ethidium bromide excretion by MaD5 and MaD3S. Fig. 7a shows the complex X-ray analysis diagram of MaD5 and PfMATE, Fig. 7b shows the chemical structure of MaD5, Fig. 7c shows the complex X-ray analysis diagram of MaD3S and PfMATE, and Fig. 7d shows the chemical structure of MaD3S.

The peptide according to the present invention inhibits the multidrug efflux transport activity of MATE protein, and has a cyclic part consisting of 3 to 15 amino acids and a tail of 1 or more amino acids.
In this specification, the MATE protein refers to a protein consisting of about 450 amino acid residues described in Non-Patent Documents 1 and 2 and having 12 transmembrane helices, and its family, including homologs thereof.
MATE protein is, for example, PfMATE derived from Pyrococcus furiosus, T. sibiricus MATE, T. naphthophila MATE, T. maritima TM1701, M. jannaschii MJ0709, M. smithii NorM, E. coli c2456, V. cholerae VcmN, V. cholerae Examples include, but are not limited to, NorM, Homosapiens MATE, homologs in mammals (such as SLC47A), and homologs in plants.
As described above, the MATE protein transports sodium ions, protons, and drugs counter to each other, and discharges drugs in cells.

In the present specification, “inhibition of multidrug efflux transport activity” means that all or part of the activity of effluxing and transporting a drug possessed by MATE protein to the outside of a cell is suppressed. Whether or not a certain peptide inhibits the multidrug efflux transport activity of MATE protein can be confirmed by those skilled in the art, for example, by the method described in Examples.
When the peptide according to the present invention was co-crystallized with MATE protein and X-ray crystal structure analysis was performed, it was confirmed that the cyclic portion was fitted in the groove of the substrate binding site of MATE protein. This suggests that the peptide according to the present invention inhibits the multidrug efflux transport activity by inhibiting the conformational change of the MATE protein. In addition, the tail portion is considered to prevent the peptide once fitted in the groove from coming off the groove.
However, the peptides according to the present invention widely include those that inhibit multidrug efflux transport activity regardless of the mechanism.

  In the peptide according to the present invention, the cyclic portion consists of 3 to 15 amino acids. Amino acids for forming a ring are also included in the 3 to 15 amino acids. The cyclic moiety may be any number of amino acids from 3 to 15, but is preferably in the range of, for example, 3 to 10, and 4 to 7.

In this specification, “amino acid” is used in its broadest sense, and includes artificial amino acid variants and derivatives in addition to natural amino acids. As used herein, amino acids include natural proteinaceous L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; natural non-protein amino acids such as norleucine, β-alanine, ornithine; and amino acid characteristics. And chemically synthesized compounds having properties known in the art. Examples of non-natural amino acids include α-methyl amino acids (such as α-methylalanine), D-amino acids, histidine-like amino acids (such as β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine and α-methyl-histidine) Amino acids having an extra methylene in the side chain ("homo" amino acids) and amino acids in which the carboxylic acid functional amino acids in the side chain are replaced with sulfonic acid groups (such as cysteic acid).
In the present specification, amino acids are represented by one letter or three letters.

As shown in the examples described later, the cyclic portion of the peptide according to the present invention is based on the sequence of the D5 peptide fitted to the substrate binding site of the MATE protein, etc., for example, the following properties (i) to (iii): It can be provided.
(i) The N-terminus is F, Y, W, H, or an enantiomer thereof, and is bonded to C at the C-terminus to form a ring.
(ii) At least two amino acids are hydrophobic amino acids.
(iii) 1 or 2 Y is included.
F, Y, W, and H are aromatic ring amino acids. In the present specification, the hydrophobic amino acid refers to A, V, L, I, M, W, F, and P.

In addition, the cyclic portion of the peptide according to the present invention is preferably 7 amino acids, and in this case, it may have at least one of the following characteristics (a) to (f).
(a) The N-terminus is F, Y, W, H, or an enantiomer thereof, and is bonded to C at the C-terminus to form a ring.
(b) The second from the N-terminus is V, L, or T.
(c) The third from the N-terminus is F, Y, or H.
(d) The fourth from the N-terminus is S, A, P, N, or Q.
(e) The fifth from the N-terminus is A, V, S, L, or T.
(f) The sixth from the N-terminus is A, V, F, or Y.
F, Y, W, and H are aromatic ring amino acids, and V, L, and T are amino acids having relatively small hydrophobic residues. F, Y, and H are all aromatic ring amino acids, and S, A, P, N, and Q are relatively small hydrophobic or neutral residues. A, V, S, L, and T are hydrophobic residues, and A, V, F, and Y are hydrophobic or aromatic ring residues.
Each amino acid may be modified, for example, an N alkyl amino acid or an amino acid whose side chain is not proteinaceous.

The cyclic portion of the peptide according to the present invention may include a sequence with VYSAV, or may include a sequence in which one amino acid is deleted, added, or substituted in the sequence with VYSAV.
When one amino acid is substituted, the place of substitution is not particularly limited, but the resulting peptide preferably has at least one of the above properties (b) to (f).
Any amino acid necessary for cyclization can be bound to the N-terminal side or C-terminal side of VYSAV.

The tail portion of the peptide according to the present invention is not particularly limited as long as it contains one or more amino acids, but preferably has a high hydrophobicity. A peptide having a highly hydrophobic tail or a tail containing 8 amino acids or more is considered to be difficult to remove from the MATE protein.
Accordingly, the tail portion preferably has at least one of the following properties (I) or (II).
(I) 30% or more of the amino acids are hydrophobic amino acids; and
(II) Including a site where two or more amino acids are continuous with hydrophobic amino acids.

Non-limiting examples of peptides according to the invention include the following:
FVYSAVCLYVGSLYPCG;
FLYNAYCLWLAYCVNSCG;
FTHPIFCYPSADLCG;
FTYSAFCYAIANIAYCG;
fQWQCHIFTNLALTCG;
fVYSAVCLYVGSLYSCG;
fVYSAVCLYVGSLYSG;
fLYNAYCLWLAYCVNSCG;
fVYSAVCYSIAAAAAAARTG;
fVYSAVCYSIAAAAAAA;
fVDASACSFVNLWLTCG;
fSVACSAFVRIAHHASCG;
fTTYSAFCYAIANIAYCG; and
fTYSAFCYAIANIAYCG

These peptides are presumed to have high multidrug efflux transport activity based on the sequence selected from the D5 peptide fitted to the substrate binding site of the MATE protein from those selected in the selection performed in the examples described later. And those with a high frequency of selection.
In the sequence, F represents L-phenylalanine, and f represents D-phenylalanine. F or f may be N-chloroacetylated, and in this case, the chloroacetyl group and C located closest to the C-terminal may be bonded to form a ring.

In the peptide according to the present invention, the C-terminus may be not only a carboxyl group or a carboxylate group, but also an amide or ester. The polypeptide of the present invention also includes a salt of the polypeptide. As a salt of a polypeptide, a salt with a physiologically acceptable base or acid is used. For example, an addition salt of an inorganic acid (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.) Addition salt of organic acid (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), inorganic base (ammonium hydroxide or alkali Or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.) and amino acid addition salts.
The polypeptide of the present invention may be fused with a peptide having a specific function so that the polypeptide is introduced into a cell when administered.
In addition, the polypeptide of the present invention is a polypeptide to which modifications such as phosphorylation, methylation, acetylation, adenylylation, ADP ribosylation, and glycosylation are added as long as the problem of the present invention is solved. May be. It may be fused with other peptides or proteins.

  The peptide according to the present invention may be produced by chemical synthesis such as liquid phase method or solid phase method, or may be produced by translational synthesis. A person skilled in the art can produce the peptide of the present invention by various methods by a known method or a method analogous thereto.

In the solid-phase method, for example, the hydroxyl group of a resin having a hydroxyl group is esterified with the carboxy group of the first amino acid (usually the C-terminal amino acid of the target peptide) in which the α-amino group is protected with a protecting group. . As the esterification catalyst, known dehydration condensing agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI) can be used.
Next, the α-amino group protecting group of the first amino acid is removed, and a second amino acid in which all functional groups other than the carboxy group of the main chain are protected is added to activate the carboxy group. , Combining the first and second amino acids. Further, the α-amino group of the second amino acid is deprotected, a third amino acid in which all functional groups other than the carboxy group of the main chain are protected is added, the carboxy group is activated, and the second and Bind a third amino acid. This is repeated, and when a peptide having a desired length is synthesized, all functional groups are deprotected.

Resins for solid phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEGA resin (Merck) Is mentioned. These resins may be used after being washed with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.).
Protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, allyloxycarbonyl (Alloc ) Group and the like. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine.
For protecting the α-carboxy group, methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester and the like can be used.
As other functional groups of amino acids, the hydroxy group of serine or threonine can be protected with a benzyl group or a tert-butyl group, and the hydroxy group of tyrosine is protected with a 2-bromobenzyloxycarbonyl dilute or tert-butyl group. The amino group of the lysine side chain and the carboxy group of glutamic acid or aspartic acid can be protected in the same manner as the α-amino group and α-carboxy group.

The activation of the carboxy group can be performed using a condensing agent. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC or WSC), and (1H-benzotriazol-1-yloxy) tris. (Dimethylamino) phosphonium hexafluorophosphate (BOP), 1- [bis (dimethylamino) methyl] -1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU) and the like.
The peptide chain can be cleaved from the resin by treatment with an acid such as TFA or hydrogen fluoride (HF).

  Production of a peptide by a genetic recombination method or a cell-free translation system can be performed using a nucleic acid (DNA or RNA) encoding the peptide of the present invention.

  The nucleic acid encoding the peptide of the present invention can be prepared by a known method or a method analogous thereto. For example, it can be synthesized by an automatic synthesizer. In order to insert the obtained DNA into a vector, a restriction enzyme recognition site may be added, or a base sequence encoding an amino acid sequence for cutting out the resulting peptide chain with an enzyme or the like may be incorporated.

  The nucleic acid encoding the peptide of the present invention can be expressed by inserting it downstream of the promoter of the expression vector. Examples of vectors include Escherichia coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast-derived plasmids ( pSH19, pSH15, YEp, YRp, YIp, YAC, etc., bacteriophage (λ phage, M13 phage, etc.), virus (retrovirus, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus , Baculovirus, etc.), cosmids, etc. can be used.

The promoter can be appropriately selected depending on the type of host. When the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or a promoter derived from CMV (cytomegalovirus) can be used. When the host is E. coli, trp promoter, T7 promoter, lac promoter, etc. can be used.
Expression vector encodes DNA replication origin (ori), selection marker (antibiotic resistance, auxotrophy, etc.), enhancer, splicing signal, poly A addition signal, tag (FLAG, HA, GST, GFP, etc.) It is also possible to incorporate nucleic acid or the like.

  An expression vector containing a nucleic acid encoding a peptide according to the present invention may be used for transforming a host in vitro as described below to express the peptide of the present invention, and can be directly administered to a subject. It can also be used to express the peptides of the invention in vivo.

The host used for transformation can be appropriately selected in relation to the vector, and for example, Escherichia coli, Bacillus subtilis, Bacillus sp.), Yeast, insect or insect cells, animal cells and the like are used. As animal cells, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells can be used. Transformation can be performed according to a known method such as a lipofection method, a calcium phosphate method, an electroporation method, a microinjection method, or a particle gun method, depending on the type of host.
The desired peptide is expressed by culturing the transformant according to a conventional method. The medium is appropriately selected according to the type of host.

For peptide purification from transformant cultures, cultured cells are collected, suspended in an appropriate buffer, disrupted by sonication, freeze-thawing, etc., and roughly extracted by centrifugation or filtration. Obtain a liquid. When the peptide is secreted into the culture solution, the supernatant is collected.
Purification from a crude extract or culture supernatant is a known method or a method equivalent thereto (for example, salting out, dialysis method, ultrafiltration method, gel filtration method, SDS-PAGE method, ion exchange chromatography, affinity chromatography, Reversed phase high performance liquid chromatography).
The obtained peptide may be converted from a free form to a salt or from a salt to a free form by a known method or a method analogous thereto.

The translation synthesis system may be a cell-free translation system. Cell-free translation systems include, for example, ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF) elongation factor (EF), termination factor (RF), and ribosome Includes regeneration factor (RRF), as well as other factors required for translation. In order to increase the expression efficiency, an Escherichia coli extract or a wheat germ extract may be added. In addition, a rabbit erythrocyte extract or an insect cell extract may be added.
By supplying energy continuously to a system containing these using dialysis, a protein of several hundred μg to several mg / mL can be produced. In order to perform transcription from DNA together, a system containing RNA polymerase may be used. As cell-free translation systems on the market, E. coli-derived systems such as RTS-100 (registered trademark) from Roche Diagnostics, PURESYSTEM (registered trademark) from PGI, etc. For example, those from Zoigene or Cell Free Science can be used.
According to the cell-free translation system, the expression product can be obtained in a highly purified form without purification.

In the cell-free translation system, an artificial aminoacyl tRNA in which a desired amino acid or hydroxy acid is linked (acylated) to tRNA may be used instead of aminoacyl tRNA synthesized by natural aminoacyl tRNA synthetase. Such aminoacyl-tRNA can be synthesized using an artificial ribozyme.
Such ribozymes include flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; H. Murakami, D. Kourouklis, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 1077-1084; H. Murakami, A. Ohta, H. Ashigai, H. Suga (2006) Nature Methods 3, 357-359 "The flexizyme system: a highly flexible tRNA aminoacylation tool for the synthesis of nonnatural peptides "; N. Niwa, Y. Yamagishi, H. Murakami, H. Suga (2009) Bioorganic & Medicinal Chemistry Letters 19, 3892-3894" A flexizyme that selectively charges amino acids activated by a water-friendly leaving group "; and WO2007 / 066627). Flexizyme is also known by the name of the original flexizyme (Fx) and dinitrobenzyl flexizyme modified from this (dFx), enhanced flexizyme (eFx), amino flexizyme (aFx) and the like.

By using tRNA with a desired amino acid or hydroxy acid generated by flexizyme, the desired codon can be associated with the desired amino acid or hydroxy acid and the genetic code can be reprogrammed and translated. Special amino acids may be used as the desired amino acids.
Examples of special amino acids are shown in the table below, but are not limited thereto. In the table, DBE and CME are ester types when a special amino acid is bound to tRNA by flexizyme, DBE means 3,5-dinitrobenzyl ester, and CME means cyanomethyl ester.

  The method for forming a cyclized structure in the peptide according to the present invention is not particularly limited, and is a disulfide bond, peptide bond, alkyl bond, alkenyl bond, ester bond, thioester bond, ether bond, thioether bond, phosphonate ether bond, azo bond, CSC bond, CNC bond, C = NC bond, amide bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, amine bond, thioamide bond, and the like can be used, but not limited thereto.

For cyclization, an amino acid chloroacetylated at the N-terminus may be used, and Cys may be arranged in the sequence. As a result, the peptide spontaneously cyclizes due to the thioether bond between the N-terminal amino acid and Cys. The thioether bond formed between the chloroacetylated amino acid and Cys is less susceptible to degradation under reducing conditions in vivo, so it is possible to increase the blood half-life of the peptide and sustain the bioactive effect. .
Examples of chloroacetylated amino acids include N-Chloroacetyl-L-alanine, N-Chloroacetyl-L-phenylalanine, N-Chloroacetyl-L-tyrosine, N-Chloroacetyl-L-tryptophan, N-Chloroacetyl-D-alanine, N -Chloroacetyl-D-phenylalanine, N-Chloroacetyl-D-tyrosine, N-Chloroacetyl-D-tryptophan, N-3-chloromethylbenzoyl-L-tyrosine, N-3-chloromethylbenzoyl-L-tryptophane can be used.
If Nγ- (2-chloroacetyl) -α, γ-diaminobutylic acid or Nγ- (2-chloroacetyl) -α, γ-diaminopropanoic acid is used as the chloroacetylated amino acid, it is introduced into any part of the peptide chain. As a result, a thioether bond is generated between the amino acid at an arbitrary position and cysteine in the same peptide, and a cyclic structure is formed.
Cyclization methods are described, for example, by Kawakami, T. et al., Nature Chemical Biology 5, 888-890 (2009); Yamagishi, Y. et al., ChemBioChem 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); Kawakami T. et al, Chemistry & Biology 15, 32- 42 (2008).

When synthesizing by cell-free translational synthesis using the reprogramming of the genetic code described above, N-chloroacetyl-L-phenylalanine or N-chloroacetyl-D-phenylalanine is charged to the starting tRNA ^fMet _CAU By inserting a cysteine at the position of, a ring is spontaneously formed after translation.

  The nucleic acid of the present invention is a nucleic acid encoding the peptide of the present invention, and may be DNA or RNA. The nucleic acid of the present invention can be prepared by a known method or a method analogous thereto. For example, it can be synthesized by an automatic synthesizer. In order to insert the obtained DNA into a vector, a restriction enzyme recognition site may be added, or a base sequence encoding an amino acid sequence for cutting out the resulting peptide chain with an enzyme or the like may be incorporated.

Since the peptide according to the present invention inhibits the multidrug excretion / transport activity of the MATE protein, it can be used to prevent adverse effects caused by the drug being excreted by the multidrug excretion / transport activity. For example, it is considered that it can be administered together with an anticancer agent, and the anticancer agent can be prevented from being excreted from cancer cells by MATE protein, and the effect of the anticancer agent can be enhanced. In addition, it is expected that it can be administered together with a therapeutic agent for multidrug-resistant bacterial infections such as MRSA to prevent the therapeutic agent from being discharged from the bacterium and enhance the effect of the therapeutic agent.
Therefore, this invention also provides the therapeutic effect enhancer of the anticancer agent or the therapeutic agent for multidrug-resistant bacteria infection containing the peptide which concerns on this invention.

As the therapeutic effect enhancer according to the present invention, the peptide according to the present invention may be used as it is, or may be formulated by adding a pharmaceutically acceptable carrier, excipient, additive and the like. Examples of the dosage form include liquids (for example, injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, Examples include inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.
Formulation includes, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjustments It can be carried out by a conventional method using an agent, preservative, antioxidant and the like as appropriate.
When the pharmaceutical composition contains a peptide, surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin are used as an absorption accelerator for improving absorption of a poorly absorbable drug that is difficult to be absorbed through the mucosa such as a peptide. Bile salts such as glycocholic acid, deoxycholic acid and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid and mixed micelles; enamine derivatives N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, nitric oxide donors, and the like can be used.

The dose of the therapeutic effect enhancer according to the present invention, and those skilled in the art can appropriately determine the dose according to the patient's symptoms, age, sex, weight, sensitivity difference, administration method, administration interval, formulation type, and the like. .
The disclosures of all patent and non-patent documents cited herein are hereby incorporated by reference in their entirety.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to this at all. Those skilled in the art can change the present invention into various modes without departing from the meaning of the present invention, and such changes are also included in the scope of the present invention.

1. Preparation of N-ClAc-L-Phe- tRNA fMet CAU and N-ClAc-D-Phe- tRNA fMet CAU
N-chloroacetyl-L-phenylalanine according to the method of Goto et al. (Goto Y. et al., Nat Protoc 2011, 6: 779-790; Goto Y. et al., ACS Chem Biol 2008, 3: 120-129) And N-chloroacetyl-D-phenylalanine were charged to the starting tRNA ^fMet _CAU . The tRNA for round 1 of the charged selection was stored at −80 ° C. as a 5.25 nmol aliquot as a dry pellet. For tRNA for round 2 and subsequent rounds, 175 pmol aliquots were stored as dry pellets at -80 ° C.

2. Library construction
The DNA library was constructed according to the method of Hayashi et al. (Hayashi, Y. et al., ACS Chemical Biology 2012, 7: 607-613). Oligonucleotide primers encoding 7-15 random amino acids were used with extension and PCR primers (table below). Complementary regions are shown in italics and identical regions are shown in bold.
Add the mRNA library to the library template NNK15: NNK14: NNK13: NNK12: NNK11: NNK10: NNK9: NNK8: NNK7 at 160: 40: 10: 2.5: 0.625: 0.156: 0.391: 0.00977: 0.00244 pmol respectively The total volume was 100 μL.

3. In round 1 of protein immobilization selection, Invitrogen Dynabeads® in HEPES buffered saline (25 mM HEPES, 150 mM NaCl, 0.1% Cymal-6 (w / v), pH 7.0, 150 (l)) ) His-Tag Isolation & Pulldown magnetic beads (15 (l) His tag binding sites were saturated using PfMATE (751 pmol) with a His tag before removing the storage buffer).
In round 2 and subsequent rounds, Invitrogen Dynabeads® in HEPES buffered saline (25 mM HEPES, 150 mM NaCl, 0.1% Cymal-6 (w / v), pH 7.0, 150 (l)) His-Tag Isolation & Pulldown magnetic beads (1 (l) His tag binding sites were saturated with His-tagged PfMATE (50 pmol) before removing the storage buffer.
The protein-bead mixture was incubated for 20 minutes at room temperature with gentle agitation. After incubation, the beads were washed 3 times with HEPES buffered saline (300 μl for round 1, 30 μl for round 2 and later) to remove unbound PfMATE.

4). mRNA-puromycin
4-1. Round 1
In round 1, ligation reaction solution (20% DMSO, 1x T4 RNA ligase buffer, 1.5 (M puromycin linker, 1 (M mRNA, 236 pmol T4 ligase, 200 (l)) was incubated at room temperature for 30 minutes. The solution was stopped by adding a solution (0.6 M NaCl, 10 mM EDTA, pH 7.5, 200 (l). The solution was extracted with 25: 24: 1 phenol: chloroform: isoamyl alcohol (400 (l)) The second extraction was performed with chloroform: isoamyl alcohol (400 (l). 800 μl of ethanol was added to the aqueous phase to precipitate the mRNA-puromycin complex. The resulting precipitate was centrifuged (13000 rpm, 15 Min.), Washed with 70% ethanol (400 μl), centrifuged (13000 rpm, 3 min) and air-dried The precipitate was dissolved in 30 μl water.

4-2. For round 2 and subsequent rounds and all subsequent rounds, ligation reaction solution (20% DMSO, 1x T4 RNA ligase buffer, 1.5 μM puromycin linker, 1 (M mRNA, 47.2 pmol T4 ligase, 40 (l)) at room temperature Incubated for 30 minutes with 40 μl of water added to the reaction solution and stopped by adding 80 μl of stop solution (0.6 M NaCl, 10 mM EDTA, pH 7.5). Extraction with phenol: chloroform: isoamyl alcohol (160 (l) and second extraction with 24: 1 chloroform: isoamyl alcohol (160 (l). Add 320 μl of ethanol to the aqueous phase and add mRNA-Puro) The mycin complex was precipitated and the precipitate was washed with 70% ethanol and air dried, and the precipitate was dissolved in 8 μl of water.

5. Selection of PfMATE-linked cyclic peptides
5-1.4 Selection at 4 ° C
Round 1 of in vitro selection was performed using 180 pmol mRNA-puromycin complex and 5.25 nmol ClAc-L-Phe-tRNA ^fMet _CAU or ClAc-D-Phe-tRNA ^fMet _CAU with a reaction volume of 150 μl. (Hayashi, Y. et al., ACS Chemical Biology 2012, 7: 607-613; Yamagishi Y. et al., Chem Biol 2011, 18: 1562-1570, schematically shown in FIGS. 1A-C).
The translation reaction solution was incubated at 37 ° C. for 30 minutes and then incubated at room temperature for 12 minutes. EDTA solution (200 mM, 15 μl) was added to each translation reaction. In order to promote circularization, the reaction solution was incubated again at 37 ° C. for 30 minutes.
The reaction solution was passed through a Sephedex G-25 column pre-washed with HPES buffered saline to remove salts. Each of the peptide-mRNA solutions excluding salt was mixed with 2x blocking solution (1 M NaCl, 0.2% acetylated bovine serum albumin, 25 mM HEPES, 150 mM NaCl, 0.1% Cymal-6 (w / v), pH 7.0, 165 (l) was added.

Components of the FIT system with unwanted bead-bound peptide-mRNA and His tag were removed by incubating the peptide-mRNA with magnetic beads (150 μl before removing the storage buffer) excluding the storage buffer.
The supernatant was separated, and 1 μl of the supernatant was used for reverse transcription reaction and real-time PCR, and the concentration of input peptide-mRNA was measured.
Of the remaining supernatant (329 μl), 300 μ of the peptide-mRNA solution was incubated with PhMATE-beads (prepared in the “Protein Immobilization” section) for 1 hour at 4 ° C. with gentle agitation.
After incubation, the supernatant was removed, and the magnetic beads were washed three times with ice-cold HEPES buffered saline (800 μl) to remove peptide-mRNA not bound to the magnetic beads. A reverse transcription reaction solution (1.25x MMLV RT buffer, 0.625 mM dNTP, 3.125 (M primer CGS3an13.R39, 8 U RNase In, 300 U RTase (+ H), 40 (l) was added to the beads. Was incubated for 1 hour at 42 ° C. Into the reverse transcription reaction containing beads, PCR reaction MIX (1x Taq PCR Buffer, 2.5 mM MgCl ₂ , 0.5 mM dNTP, 0.25 (M primer T7g10M.F48) without polymerase was added. , 0.25 (M primer GS3an13.R39) and incubated for 5 minutes at 95 ° C. Transfer the supernatant to a new tube and add 600 μl polymerase-free PCR Reaction MIX and 6 μl Taq. In order to quantify the recovered peptide-mRNA, 20 μl was removed from the reaction solution for real-time PCR.The remaining 826 μl of solution was 16 × (95 ° C./40 seconds, 50 ° C./40 seconds, 72 ° C./40 The PCR product was subjected to one equivalent volume of 25: 24: 1 phenol: chloroform: isoamid. And extracted with alcohol, followed by 1 equivalent of 24: 1 chloroform: extracted with isoamyl alcohol.
NaCl solution (3M, 82 μl) was added to the aqueous phase, and ethanol (1640 μl) was added to precipitate the DNA product. The pellet was dissolved in KCl solution (50 mM, 30 μl). Dissolved DNA (4 μl) was added to 16 μl of a transcription reaction solution (1.2 × T7 buffer, 12 mM dithiothreitol, 24 mM MgCl ₂ , 26.4 mM KOH, 4.5 mM NTPs, 4 U RNase inhibitor, and T7 polymerase). . The transcription reaction solution was incubated overnight (12 hours or longer) at 37 ° C.

Rounds 2 to 7 were selected with modifications to the above method.
In vitro translation reaction was performed using 175 pmol of ClAc-L-Phe-tRNA ^fMet _CAU or ClAc-D-Phe-tRNA ^fMet _CAU and 7.5 pmol of mRNA-puromycin complex (total amount: 5 μl). To prevent isolation of RNA aptamers with affinity for the target protein, complementary DNA was reverse transcribed prior to selection.
Selection incubation was reduced to 30 minutes. While melting DNA from peptide-mRNA, PCR Reaction MIX without 100 μl of polymerase was used. The amount of peptide-mRNA bound to the beads was determined by taking a 1 μl aliquot from the supernatant containing single stranded DNA.
No further enrichment was seen after round 7 (Figure 2A). The seventh generation DNA library was ligated to the pGEM-T Easy plasmid using TA-cloning. Arbitrary individual clones were picked and sequenced by FASMAC. The respective sequences are shown in the table below.

5-2. Selection at 37 ° C. The second selection was performed in the same manner as “Selection at 4 ° C.” except that the selection and washing steps of all beads were performed at 37 ° C. along with phMATE fixation. Also, propargyl and ethylindolyl N-substituted glycine-tRNA (Kawakami et al., J Am Chem Soc 2008, 130: 16861-16863) were used to fill empty codons CAU (extension) and UAA (amber), respectively. Using. However, the finally selected clones did not contain monomer units. Furthermore, in Round 6 of this second selection performed at 37 ° C., selection was performed under two conditions. In one of the conditions of Round 6, chemically synthesized MATEin-L6 (final concentration 32 μM, which will be described later) without mRNA was added. The other selection criteria followed the previous round. Selection was completed because sufficient enrichment was seen in round 6 (FIG. 2B).
The sixth generation obtained from this selection was cloned and sequenced, with or without the competitive binding peptide MATEin-L6 (Table 2).
The clonal assay was performed as described in “Selection at 4 ° C.”. MATEin-D1 and MATEin-D5 were synthesized with an automatic synthesizer (Syro, Biotage).

6). Clonal assay The binding ability of individual clones was examined using mRNA transcribed from individual clones instead of library mRNA (Figure 2C). The conditions of the assay were the same as those after round 2 of the selection.

7). Chemical synthesis of peptides Peptides were chemically synthesized by replacing the (Gly-Ser) ₃ linker used in the selection with only one glycine. MATEin-L6 was synthesized manually. MATEin-D8 was synthesized with an automatic peptide synthesizer (Syro, Biotage). The peptide is cleaved from the resin and then subjected to the method of Yamagishi or Hayashi et al. (Yamagishi Y. et al., Chem Biol 2011, 18: 1562-1570; Hayashi, Y. et al., ACS Chemical Biology 2012, 7: 607- 613). The cyclic peptide was purified by HPLC (Imtakt Cadenza CD-C18 250 × 10 mm column, Gilson HPLC). Mass was confirmed with an MLDI-ROF mass spectrometer (autoflex speed, Bruker) (FIG. 3).

8). Inhibition assay
Inhibitory activity of MATEin-L6, -D1, -D5, and -D8 peptides using an ethidium accumulation method modified from the method of Li et al. (Li X et al., Antimicrob Agents Chemother, 47: 27-33) Examined. PfMATE was expressed using E. coli ΔAcrB. Cells expressing PfMATE were cultured overnight in LB medium containing 100 mg / l ampicillin at 37 ° C. with shaking at 200 rpm. Formal E. coli was diluted 100 times (volume) with fresh LB medium containing 100 mg / l ampicillin. The cells were cultured until OD ₆₀₀ reached 0.5, and IPTG (final concentration 0.5 mM) was added to induce expression. Induced cells were incubated overnight at 20 ° C. with shaking at 200 rpm. The cells were washed once with 0.5 M Tris buffer (pH 7.0) and resuspended in 0.5 M Tris buffer (pH 7.0) to a final OD ₆₀₀ of 0.5.
In each well of a 96-well black plate (1/2 area, Perkin Elmer), 44.5 μl of the cell suspension was added to 0-250 μM 0.5 μl peptide (in DMSO). As a positive control, carbonyl cyanide-m-chlorophenylhydrazone (final concentration 100 μM) was added to one sample. Ethidium bromide (0.5 mM, 5 μl) was added to a final concentration of 50 μM, and the plate was immediately placed on the plate reader Flexastation 3. It took approximately 15 seconds from the addition of ethidium bromide to the first reading (t = 0 seconds) (Molecular Devices).
The results are shown in FIG.
It was confirmed that all of the D1, D5, and D8 peptides inhibited the multidrug efflux transport activity of MATE protein in a dose-dependent manner. In particular, D5 showed a high inhibitory effect. On the other hand, L6 showed no inhibitory activity.
CCCP (Carbonyl cyanide m-chlorophenylhydrazone) is a MATE inhibitor, and the data of CCCP shows the case where 100% inhibition is caused by chemically losing the function of PhMATE.

9. X-ray crystal structure analysis MaD5 and MaD3S having the sequence shown in Fig. 5a were synthesized, co-crystallized with PfMATE, respectively, and complex X-ray structure analysis was performed. The result is shown in FIG. The peptide in the tail portion could not be visualized by the complex X-ray structure analysis due to its dynamic state, but is expected to be outward from the active site of PfMATE in an α-helix shape. In the figure, the peptides are visualized with a space filling model.
The inhibitory activity of these peptides is also described in the above 8. Similarly, the amount of ethidium bromide (EtBr) accumulated inside was detected. The actual measurement data is shown in FIG. 6, and the result of the concentration of 1 μM is shown in FIG. 5c. The control indicates the amount of ethidium bromide that remains in the cell without being excreted outside the cell when PfMATE is fully functioning.
FIG. 7a shows a complex X-ray analysis diagram of MaD5 and PfMATE. Amino acids shown by dotted lines are invisible sites in structural analysis. FIG. 7b is a chemical structure of MaD5, and FIG. 7c is a complex X-ray analysis diagram of MaD3S and PfMATE. Amino acids shown by dotted lines are invisible sites in structural analysis. FIG. 7d shows the chemical structure of MaD3S.
As shown in FIGS. 5 to 7, the peptide according to the present invention was fitted in the groove of the substrate binding site of the MATE protein and inhibited the excretion function.
When L6 was co-crystallized with MATE protein and X-ray structural analysis was performed, it was confirmed that L6 peptide did not fit into the groove of the substrate binding site of MATE protein and was attached to the outer surface of the protein. (Data not shown). This is because L6 is the above 8. This is consistent with the fact that no inhibition activity against MATE was shown in the inhibition assay.

Claims (3)

Any of the following peptides:
FVYSAVCLYVGSLYPCG;
FLYNAYCLWLAYCVNSCG;
FTHPIFCYPSADLCG;
FTYSAFCYAIANIAYCG;
fQWQCHIFTNLALTCG;
fVYSAVCLYVGSLYSCG;
fVYSAVCLYVGSLYSG;
fLYNAYCLWLAYCVNSCG;
fVYSAVCYSIAAAAAAARTG;
fVYSAVCYSIAAAAAAA;
fVDASACSFVNLWLTCG;
fSVACSAFVRIAHHASCG;
fTTYSAFCYAIANIAYCG; and
fTYSAFCYAIANIAYCG
(In the formula, F at the N-terminal represents L-phenylalanine and f represents D-phenylalanine. F or f is N-chloroacetylated, and the chloroacetyl group is bonded to C at the most N-terminal side to form a ring. to form a.).   A nucleic acid encoding the peptide according to claim 1.   The therapeutic effect enhancer of the anticancer agent or the multidrug resistant bacterium infection therapeutic agent containing the peptide of Claim 1.