Methods and compositions for ribosomal synthesis of macrocyclic peptides
First Claim
Patent Images
1. A method for making a macrocyclic peptide, the method comprising:
- (a) providing an artificial nucleic acid molecule encoding for a polypeptide of structure;
(AA)m-Z-(AA)n-Cys-(AA)p
(I)
or
(AA)m-Cys-(AA)n-Z-(AA)p
(II)
or
(AA)m-Cys-(AA)n-Z2-(AA)o-Cys-(AA)p
(V)wherein;
(i) (AA)m is an N-terminal amino acid or peptide sequence,(ii) Z is a non-canonical amino acid carrying a side-chain functional group FG1, FG1 being a functional group selected from the group consisting of —
(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10;
—
C(O)CH2X, where X is F, Cl, Br, or I;
—
CH(R′
)X, where X is F, Cl, Br, or I;
—
C(O)CH(R′
)X, where X is F, Cl, Br, or I;
—
OCH2CH2X, where X is F, Cl, Br, or I;
—
C(O)CH═
C═
C(R′
)(R″
);
—
SO2C(R′
)═
C(R′
)(R″
);
—
C(O)C(R′
)═
C(R′
)(R″
);
—
C(R′
)═
C(R′
)C(O)OR′
;
—
C(R′
)═
C(R′
)C(O)N(R′
)(R″
);
—
C(R′
)═
C(R′
)—
CN;
—
C(R′
)═
C(R′
)—
NO2;
—
C≡
C—
C(O)OR′
;
—
C≡
C—
C(O)N(R′
)(R″
);
unsubstituted or substituted oxirane;
unsubstituted or substituted aziridine;
1,2-oxathiolane 2,2-dioxide;
4-fluoro-1,2-oxathiolane 2,2-dioxide; and
4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and
R″
is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group,(iii) Z2 is a non-canonical amino acid carrying two side-chain functional groups FG1 and FG2, wherein each of FG1 and FG2 is a functional group independently selected from the group consisting of —
(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10;
—
C(O)CH2X, where X is F, Cl, Br, or I;
—
CH(R′
)X, where X is F, Cl, Br, or I;
—
C(O)CH(R′
)X, where X is F, Cl, Br, or I;
—
OCH2CH2X, where X is F, Cl, Br, or I;
—
C(O)CH═
C═
C(R′
)(R″
);
—
SO2C(R′
)═
C(R′
)(R″
);
—
C(O)C(R′
)═
C(R′
)(R″
);
—
C(R′
)═
C(R′
)C(O)OR′
;
—
C(R′
)═
C(R′
)C(O)N(R′
)(R″
);
—
C(R′
)═
C(R′
)—
CN;
—
C(R′
)═
C(R′
)—
NO2;
—
C≡
C—
C(O)OR′
;
—
C≡
C—
C(O)N(R′
)(R″
);
unsubstituted or substituted oxirane;
unsubstituted or substituted aziridine;
1,2-oxathiolane 2,2-dioxide;
4-fluoro-1,2-oxathiolane 2,2-dioxide; and
4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and
R″
is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group,(iv) (AA)n is a target peptide sequence,(v) (AA)o is a second target peptide sequence, and(vi) (AA)p is a C-terminal amino acid or peptide sequence;
(b) introducing the nucleic acid molecule into an expression system, wherein the expression system is an in vivo cell system, and wherein the in vivo cell system comprises;
an aminoacyl-tRNA synthetase selected from the group consisting of SEQ ID NOs;
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100; and
a transfer RNA molecule encoded by a polynucleotide selected from the group consisting of SEQ ID NOs;
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, and 120; and
expressing the nucleic acid molecule in the in vivo cell system, thereby producing the polypeptide; and
(c) cyclizing the polypeptide via nucleophilic substitution of a peptide side group by a cysteine sulfhydryl group, wherein the cyclizing comprises allowing the functional group FG1, and whenever present, FG2, to react with the side-chain sulfhydryl group (—
SH) of the cysteine (Cys) residue(s) in the in vivo cell system, thereby producing the macrocyclic peptide in the in vivo cell system.
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Abstract
Methods and compositions are provided for generating macrocyclic peptides from genetically encoded, ribosomally produced polypeptide precursors. Also provided are nucleic acid molecules, polypeptides, and methods for generating combinatorial libraries of macrocyclic peptides. These methods can be used to produce vast libraries of conformationally constrained peptide ligands as well as facilitate the functional screening of these libraries to identify compound(s) with desired activity properties.
8 Citations
18 Claims
-
1. A method for making a macrocyclic peptide, the method comprising:
-
(a) providing an artificial nucleic acid molecule encoding for a polypeptide of structure;
(AA)m-Z-(AA)n-Cys-(AA)p
(I)
or
(AA)m-Cys-(AA)n-Z-(AA)p
(II)
or
(AA)m-Cys-(AA)n-Z2-(AA)o-Cys-(AA)p
(V)wherein; (i) (AA)m is an N-terminal amino acid or peptide sequence, (ii) Z is a non-canonical amino acid carrying a side-chain functional group FG1, FG1 being a functional group selected from the group consisting of —
(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10;
—
C(O)CH2X, where X is F, Cl, Br, or I;
—
CH(R′
)X, where X is F, Cl, Br, or I;
—
C(O)CH(R′
)X, where X is F, Cl, Br, or I;
—
OCH2CH2X, where X is F, Cl, Br, or I;
—
C(O)CH═
C═
C(R′
)(R″
);
—
SO2C(R′
)═
C(R′
)(R″
);
—
C(O)C(R′
)═
C(R′
)(R″
);
—
C(R′
)═
C(R′
)C(O)OR′
;
—
C(R′
)═
C(R′
)C(O)N(R′
)(R″
);
—
C(R′
)═
C(R′
)—
CN;
—
C(R′
)═
C(R′
)—
NO2;
—
C≡
C—
C(O)OR′
;
—
C≡
C—
C(O)N(R′
)(R″
);
unsubstituted or substituted oxirane;
unsubstituted or substituted aziridine;
1,2-oxathiolane 2,2-dioxide;
4-fluoro-1,2-oxathiolane 2,2-dioxide; and
4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and
R″
is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group,(iii) Z2 is a non-canonical amino acid carrying two side-chain functional groups FG1 and FG2, wherein each of FG1 and FG2 is a functional group independently selected from the group consisting of —
(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10;
—
C(O)CH2X, where X is F, Cl, Br, or I;
—
CH(R′
)X, where X is F, Cl, Br, or I;
—
C(O)CH(R′
)X, where X is F, Cl, Br, or I;
—
OCH2CH2X, where X is F, Cl, Br, or I;
—
C(O)CH═
C═
C(R′
)(R″
);
—
SO2C(R′
)═
C(R′
)(R″
);
—
C(O)C(R′
)═
C(R′
)(R″
);
—
C(R′
)═
C(R′
)C(O)OR′
;
—
C(R′
)═
C(R′
)C(O)N(R′
)(R″
);
—
C(R′
)═
C(R′
)—
CN;
—
C(R′
)═
C(R′
)—
NO2;
—
C≡
C—
C(O)OR′
;
—
C≡
C—
C(O)N(R′
)(R″
);
unsubstituted or substituted oxirane;
unsubstituted or substituted aziridine;
1,2-oxathiolane 2,2-dioxide;
4-fluoro-1,2-oxathiolane 2,2-dioxide; and
4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and
R″
is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group,(iv) (AA)n is a target peptide sequence, (v) (AA)o is a second target peptide sequence, and (vi) (AA)p is a C-terminal amino acid or peptide sequence; (b) introducing the nucleic acid molecule into an expression system, wherein the expression system is an in vivo cell system, and wherein the in vivo cell system comprises; an aminoacyl-tRNA synthetase selected from the group consisting of SEQ ID NOs;
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100; anda transfer RNA molecule encoded by a polynucleotide selected from the group consisting of SEQ ID NOs;
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, and 120; andexpressing the nucleic acid molecule in the in vivo cell system, thereby producing the polypeptide; and (c) cyclizing the polypeptide via nucleophilic substitution of a peptide side group by a cysteine sulfhydryl group, wherein the cyclizing comprises allowing the functional group FG1, and whenever present, FG2, to react with the side-chain sulfhydryl group (—
SH) of the cysteine (Cys) residue(s) in the in vivo cell system, thereby producing the macrocyclic peptide in the in vivo cell system. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
-
-
13. An isolated recombinant host cell comprising a polypeptide of structure:
-
(AA)m-Z-(AA)n-Cys-(AA)p
(I)
or
(AA)m-Cys-(AA)n-Z-(AA)p
(II)
or
(AA)m-Cys-(AA)n-Z2-(AA)o-Cys-(AA)p
(V)wherein; (i) (AA)m is an N-terminal amino acid or peptide sequence, (ii) Z is an amino acid of structure; - View Dependent Claims (14, 15, 16, 17, 18)
-
Specification