METHODS AND COMPOSITIONS FOR RIBOSOMAL SYNTHESIS OF MACROCYCLIC PEPTIDES

• US 20160355552A1
• Filed: 12/23/2014
• Published: 12/08/2016
• Est. Priority Date: 12/23/2013
• Status: Active Grant

• Current Assignee: University of Rochester
• Original Assignee: University of Rochester
• Inventors: Fasan, Rudi
• Granted Patent: US 10,544,191 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07K 1/113 without change of the primary structure
• CPC Class Codes: C07K 2319/92 containing an intein ("protein splicing")domain
• CPC Class Codes: C07K 7/04 Linear peptides containing only normal peptide links
• CPC Class Codes: C07K 7/54 with at least one abnormal peptide link in the ring
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

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.

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 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.

2. The method of claim 1 wherein Z is an amino acid of structure:

5. The method of claim 1 wherein the amino acid Z is selected from the group consisting of 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, N^ε

-((2-bromoethoxy)carbonyl)-lysine, N^ε

-((2-chloroethoxy)carbonyl)-lysine, N^ε

-(buta-2,3-dienoyl)-lysine, N^ε

-acryl-lysine, N^ε

-crotonyl-lysine, N^ε

-(2-fluoro-acetyl)-lysine, and N^ε

-(2-chloro-acetyl)-lysine.

6. The method of claim 1 wherein Z2 is an amino acid of structure:

10. The method of claim 1 wherein the codon encoding for Z or Z2 is an amber stop codon TAG, an ochre stop codon TAA, an opal stop codon TGA, or a four base codon.

11. The method of claim 1, wherein the expression system comprises:

an aminoacyl-tRNA synthetase polypeptide or an engineered variant thereof that is at least 90% identical to SEQ ID NO;

77, 78, 79, or 80; and

a transfer RNA molecule encoded by a polynucleotide that is at least 90% identical to SEQ ID NO;

101, 105, 109, 113, or 117.

14. The method of claim 1, wherein the expression 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 NO;

101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, and 120.

15. The method of claim 1, wherein the N-terminal tail polypeptide, (AA)_m, or the C-terminal tail polypeptide, (AA)_p, or both, of the precursor polypeptides of formula (I), (II), or (V) comprise(s):

a polypeptide affinity tag, a DNA-binding polypeptide, a protein-binding polypeptide, an enzyme, a fluorescent protein, an intein protein, or

a combination thereof.

20. The method of claim 1, wherein

the N-terminal tail polypeptide, (AA)_m, of the precursor polypeptide of formula (I), (II), or (V) comprises the C-domain of a split intein, and

the C-terminal tail polypeptide, (AA)_p, comprises the corresponding N-domain of the split intein.

22. The method of claim 1 wherein the expression system is selected from the group consisting of a prokaryotic cell, an eukaryotic cell, and a cell-free expression system.

23. The method of claim 22 wherein the prokaryotic cell is Escherichia coli.

24. The method of claim 22 wherein the eukaryotic cell is a yeast, a mammalian, an insect or a plant cell.

25. The method of claim 1 wherein any of polypeptides (AA)_n, (AA)_o, (AA)_m, or (AA)_p, is fully or partially genetically randomized so that a plurality of macrocyclic peptides is obtained upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG₁ in Z or between the cysteine (Cys) residues and the side-chain functional groups FG₁ and FG₂ in Z2.

26. The method of claim 1 comprising:

fully or partially randomizing any of polypeptides (AA)_n, (AA)_o, (AA)_m, or (AA)_p, wherein, upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG₁ in Z or between the cysteine (Cys) residues and the side-chain functional groups FG₁ and FG₂ in Z2, a plurality of macrocyclic peptides is produced.

3-4. -4. (canceled)

7-9. -9. (canceled)

12-13. -13. (canceled)

16-19. -19. (canceled)

21. (canceled)

27. 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;

28. The cell of claim 27, wherein the amino acid Z is selected from the group consisting of 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, N^ε

-((2-bromoethoxy)carbonyl)-lysine, N^ε

-((2-chloroethoxy)carbonyl)-lysine, N^ε

-(buta-2,3-dienoyl)-lysine, N^ε

-acryl-lysine, N^ε

-crotonyl-lysine, N^ε

-(2-fluoro-acetyl)-lysine, and N^ε

-(2-chloro-acetyl)-lysine.

29. The cell of claim 27 wherein the amino acid Z2 is selected from the group consisting of 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, N^ε

-(((1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, N^ε

-(((1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, N^ε

-((2,3-dibromopropoxy)carbonyl)-lysine, and N^ε

-((2,3-dichloropropoxy)carbonyl)-lysine.

30. The cell of claim 27, wherein the polypeptide comprised within the N-terminal tail polypeptide, (AA)_m, or the C-terminal tail polypeptide, (AA)_p, or both, of the precursor polypeptides of formula (I), (II), and (V), is a polypeptide selected from the group of polypeptides consisting of SEQ ID NOs 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, and 158.

32. The cell of claim 27, wherein the N-terminal tail polypeptide, (AA)_m, or the C-terminal tail polypeptide, (AA)_p, or both, in the precursor polypeptides of formula (I), formula (II), or formula (V) comprise(s) an intein selected from the group consisting of a naturally occurring intein, an engineered variant of a naturally occurring intein, a fusion of the N-terminal and C-terminal fragments of a naturally occurring split intein and a fusion of the N-terminal and C-terminal fragments of an engineered split intein.

36. The cell of claim 27, wherein:

the N-terminal tail polypeptide, (AA)_m, comprises the C-domain of a naturally occurring split intein, or of an engineered variant thereof, and

the C-terminal tail polypeptide, (AA)_p, comprises the N-domain of said split intein.

31. (canceled)

33-35. -35. (canceled)

37-38. -38. (canceled)