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US 2016/0355552 A1Published application
Methods and compositions for ribosomal synthesis of macrocyclic peptides
AssigneeUniversity of Rochester
InventorRudi Fasan
Life of this patent
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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.
Claims28
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 and expressing the nucleic acid molecule in the expression system, thereby producing the polypeptide;
- and
- (c) allowing the functional group FG1, and whenever present, FG2, to react with the side-chain sulfhydryl group (—SH) of the cysteine (Cys) residue(s), thereby producing the macrocyclic peptide.
49 more elements
3-4. (canceled)
7-9. (canceled)
12-13. (canceled)
16-19. (canceled)
(canceled)
A 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:
- wherein FG1 is 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;
- wherein Y is a linker group selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups,
- (iii) Z2 is an amino acid of structure:
- 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;
- and
- wherein Y2, Y3, L are linker groups selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups,
- (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.
51 more elements
(canceled)
33-35. (canceled)
37-38. (canceled)
Dependencies are inferred from the claim text.