Methods of constructing and screening libraries of peptide structures

• US 20080081768A1
• Filed: 02/07/2007
• Published: 04/03/2008
• Est. Priority Date: 02/20/2006
• Status: Active Grant

• Current Assignee: Phylogica Limited
• Original Assignee: Phylogica Limited
• Inventors: Dunbrack, Roland, Watt, Paul
• Granted Patent: US 8,575,070 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C07K 1/00 General methods for the preparation of peptides , i.e. processes for the organic chemical preparation of peptides or proteins of any length
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12N 15/1089 Design, preparation, screening or analysis of libraries using computer algorithms
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/6824 involving N-terminal degradation, e.g. Edman degradation
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 Detection of binding sites or motifs
• CPC Class Codes: G16B 20/50 Mutagenesis
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16B 35/20 Screening of libraries
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry

Abstract

The present invention provides the means for producing libraries of peptide structures for drug screening applications that are capable of folding or assuming their native conformations independently of artificial scaffolds or flanking sequences in the proteins from which they are derived. The libraries can be highly diverse such that they are representative of the repertoire of protein structures existing in nature. The libraries can also be non-redundant or normalized such that the bias towards specific structures existing in source data sets and/or in nature is/are removed. In a particularly preferred embodiment, the present invention provides 30,000 independent fold structures produced by this method. The present invention also provides computer-readable media and systems comprising structural data in relation to the peptide libraries, and methods for displaying and screening the libraries.

Claims

1. A method for producing a peptide library, said method comprising:

(i) obtaining a plurality of amino acid sequences capable of independently-forming secondary structures and/or assemblies of secondary structures and/or folds;

(ii) producing peptides having the amino acid sequences obtained at (i); and

(iii) displaying the peptides at (ii) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.

2. The method of claim 1 wherein a capability of independently-forming secondary structures and/or assemblies of secondary structures and/or folds is determined according to one or more criteria selected from compactness, non-polar buried surface area, and degree of independence.

3. The method of claim 1 wherein the amino acid sequences are capable of forming secondary structures and/or folds and the peptides are displayed so as to form secondary structures and/or folds.

4. The method of claim 1 wherein the amino acid sequences are capable of forming assemblies of secondary structures and/or folds and the peptides are displayed so as to form assemblies of secondary structures and/or folds.

5. The method of claim 1 wherein the amino acid sequences are capable of forming folds and the peptides are displayed so as to form folds.

6. The method of claim 1 wherein the secondary structures and/or assemblies of secondary structures and/or folds form autonomously.

7. The method of claim 1 wherein formation of the secondary structures and/or assemblies of secondary structures and/or folds is induced.

8. The method of claim 1 wherein the peptides mimic tertiary structures produced by interaction of non-contiguous portions of native proteins.

9. The method of claim 1 further comprising size-selecting sequences at (i) to thereby identify a sub-set of sequences having the average length of an independent protein fold.

10. The method of claim 1 further comprising identifying redundant sequences and removing or deleting redundant sequences to thereby leave a non-redundant or normalized plurality of amino acid sequences.

11. The method of claim 1 further comprising identifying related sequences to the obtained plurality of amino acid sequences and adding those sequences to the plurality of amino acid sequences.

12. The method of claim 11 wherein the related sequences are identified from a data source that is different to the source of the plurality of amino acid sequences.

13. The method of claim 1 further comprising mutating peptides that are predicted to form a secondary structure or assembly of secondary structures or fold.

14. The method of claim 1 further comprising mutating nucleic acids encoding the peptides.

15. The method of claim 13 wherein mutating of the peptides produces peptides having different affinities for particular ligands.

16. The method of claim 1 further comprising performing combinations selected from:

(i) (a) mutating peptides and (b) identifying redundant sequences and removing or deleting redundant sequences to thereby leave a non-redundant or normalized plurality of amino acid sequences.

(ii) (a) identifying related sequences to the obtained plurality of amino acid sequences and adding those sequences to the plurality of amino acid sequences and (b) identifying redundant sequences and removing or deleting redundant sequences to thereby leave a non-redundant or normalized plurality of amino acid sequences; and

(iii) (a) mutating peptides and (b) identifying redundant sequences and removing or deleting redundant sequences to thereby leave a non-redundant or normalized plurality of amino acid sequences and (c) identifying related sequences to the obtained plurality of amino acid sequences and adding those sequences to the plurality of amino acid sequences.

17. The method of claim 16 comprising performing iterations of any one of (i) and/or (ii) and/or (iii) in any order to thereby produce a non-redundant yet highly diverse data set.

18. The method of claim 1 wherein the peptides are produced by synthetic means.

19. The method of claim 1 wherein the peptides are produced by recombinant means.

20. The method of claim 1 wherein the peptides are displayed as arrays on a solid surface or a plurality of solid surfaces.

21. The method of claim 1 wherein the peptides are expressed on the surface of a phage or a cell or by ribosome display or by in vitro display or expressed within a cell or a plurality of cells.

29. The of claim 1 further comprising providing the amino acid sequences of the secondary structures and/or assemblies of secondary structures and/or folds produced.

40. A process comprising:

(i) performing the method of claim 1 to thereby produce a peptide library; and

(ii) screening the peptide library so produced to thereby identify a peptide.

41. A process comprising:

(i) obtaining a peptide library produced by the method of claim 1;

and

(ii) screening the peptide library to thereby identify a peptide.

42. The process of claim 40 further comprising isolating the identified peptide.

43. The process of claim 42 further comprising subjecting the isolated peptide to mutation or an affinity maturation.

44. The process of claim 40 further comprising identifying a mimetic of the structure formed by a peptide.

45. A process comprising:

(i) performing the method of claim 1 to thereby produce a peptide library;

(ii) screening the peptide library so produced to thereby identify a peptide having a desired structure; and

(iii) optionally, providing the peptide or the structure of the peptide to a person.

46. The process of claim 45 further comprising identifying one or more chemical compounds having the secondary structure and/or activity of the peptide.

22. A method for producing a peptide library having low structure redundancy, said method comprising:

(i) obtaining a plurality of amino acid sequences capable of independently-forming secondary structures and/or assemblies of secondary structures and/or folds;

(ii) identifying redundant structures from the plurality at (i) and removing or deleting redundant sequences capable of forming the redundant structures to thereby leave a non-redundant plurality of amino acid sequences;

(iii) producing peptides having the amino acid sequences of the non-redundant plurality at (ii); and

(iv) displaying the peptides at (iii) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.

23. The method according to claim 22 wherein the non-redundant plurality includes related structures that differ in their ability to fold autonomously.

24. The method according to claim 22 wherein the non-redundant plurality includes related structures that differ in their ligand-binding affinities and/or association/dissociation constants for a ligand.

25. The method according to claim 22 wherein the non-redundant plurality includes related structures that differ in their chemical modifications.

26. A method for producing a peptide library having low structure redundancy, said method comprising:

(i) obtaining a plurality of amino acid sequences capable of forming independent-secondary structures and/or assemblies of secondary structures and/or folds;

(ii) producing peptides having the amino acid sequences obtained at (i);

(iii) identifying redundant sequences from the peptides produced at (ii) and removing or deleting peptides having the redundant sequences to thereby leave a non-redundant plurality of amino acid sequences; and

(iv) displaying the peptides at (iii) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.

27. A method for producing a peptide library, said method comprising:

(i) identifying a plurality of amino acid sequences capable of folding independently from other parts of the proteins in which they are contained in their native contexts;

(ii) size-selecting those sequences at (i) to thereby identify a sub-set of sequences having the average length of an independent protein fold;

(iii) identifying redundant sequences from the sequences selected at (ii) and removing or deleting redundant sequences to thereby leave a non-redundant plurality of amino acid sequences;

(iv) producing peptides from the non-redundant plurality of amino acid sequences at (iii); and

(v) displaying the peptides at (iv) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.

28. A method for producing a peptide library, said method comprising:

(i) identifying a plurality of amino acid sequences capable of folding independently from other parts of the proteins in which they are contained in their native contexts;

(ii) size-selecting those sequences at (i) to thereby identify a sub-set of sequences having the average length of an independent protein fold;

(iii) identifying redundant sequences from the sequences selected at (ii) and removing or deleting redundant sequences to thereby leave a non-redundant plurality of amino acid sequences;

(iv) producing a diverse pool of sequence by a process comprising identifying related sequences to the non-redundant plurality of amino acid sequences at (iii) and adding those sequences to the non-redundant plurality of amino acid sequences at (iii);

(v) producing peptides from the diverse pool of sequences at (iv); and

(vi) displaying the peptides at (v) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.

30. A computer-readable medium for use in screening applications said computer-readable medium comprising a database of non-redundant amino acid sequences capable of forming independent folds or a selected subset of said plurality.

31. The computer-readable medium according to claim 30 wherein the subset of the plurality is selected on the basis of amino acid content or composition, pI, or a combination thereof.

32. A computer system for use in screening applications said computer system comprising a computer-readable medium comprising a database of non-redundant amino acid sequences capable of forming independent folds or a selected subset of said pluralist and a user interface allowing a user to input protein structure data and/or ligand structure data.

33. The computer system according to claim 32 wherein the subset of the plurality is selected on the basis of amino acid content or composition, pI, or a combination thereof.

34. A peptide library comprising a plurality of non-redundant amino acid sequences capable of forming independent folds or a selected subset of said plurality.

35. The peptide library according to claim 34 wherein the subset of the plurality is selected on the basis of amino acid content or composition, pI, or a combination thereof.

36. A peptide library comprising a plurality of peptides having sequences comprising SEQ ID NOs:

1-30000 or a subset thereof.

37. The peptide library according to claim 36 wherein the subset of the pluralist is selected on the basis of amino acid content or composition, pI, or a combination thereof.

38. A high-throughput system for drug screening comprising a solid support consisting essentially of or having a plurality of peptides bound directly or indirectly thereto, wherein said plurality of peptides comprises non-redundant amino acid sequences capable of forming independent folds or a subset of said plurality.

39. The high-throughput system of claim 38 wherein the subset of the pluralist is selected on the basis of amino acid content or composition, pI, or a combination thereof.