Methods of constructing and screening libraries of peptide structures

• US 8,575,070 B2
• Filed: 02/07/2007
• Issued: 11/05/2013
• Est. Priority Date: 02/20/2006
• Status: Active Grant

• Current Assignee: Phylogica Limited
• Original Assignee: Phylogica Limited
• Inventors: Watt, Paul Michael, Dunbrack, Roland
• Primary Examiner(s): Steele, Amber D
• Application Number:
  US11/672,419
• Publication Number:
  US 20080081768A1
• Time in Patent Office: 2,463 Days
• Field of Search: 506/4, 506/5, 506/6, 506/9, 506/24, 506/25
• US Class Current: 506/25
• 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 library of independent protein secondary structures, said method consisting essentially of the following sequential steps:

(i) executing a computer program to thereby obtain a plurality of amino acid sequences of different proteins from bioinformatic data source(s) wherein the sequences are predicted to form secondary structures and/or assemblies of secondary structures in their native contexts;

(ii) executing a computer program to simultaneously select from (i) a plurality of amino acid sequences of the different proteins, wherein the simultaneously-selected sequences each consist of a single segment of a protein corresponding to a hydrophobic folding unit is predicted to form a secondary structures independent of any other part of the protein from which it is derived when isolated from those other parts of the protein;

(iii) chemically-synthesizing a plurality of peptides that each consist essentially of a sequence selected at (ii) that is predicted to form a secondary structure independent of any other part of the protein from which it is derived when isolated from other parts of the protein; and

(iv) displaying a plurality of chemically-synthesized peptides that each consists essentially of a sequence selected at (iii) that is predicted to independently-form a secondary structure such that the displayed peptides form secondary structures independent of other parts of the proteins from which they are derived, thereby producing a peptide library of independent protein secondary structures.

2. The method of claim 1 wherein said method further comprises predicting that the plurality of different proteins will independently-form secondary structures and/or assemblies of secondary structures by determining 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 predicted to form secondary structures and wherein the peptides having said sequences are displayed so as to form secondary structures.

4. The method of claim 1 wherein the secondary structures of the displayed peptides form autonomously.

5. The method of claim 1 wherein formation of the secondary structures is induced.

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

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

8. 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.

9. 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.

10. The method of claim 9 wherein the related sequences are identified from a data source that is different to the bioinformatic data source(s) of the plurality of amino acid sequences.

11. The method of claim 1 further comprising mutating peptides having sequences that are predicted to form secondary structures.

12. The method of claim 11 wherein mutating of the peptides produces peptides having different affinities for particular ligands.

13. The method of claim 1 further comprising producing and mutating nucleic acids encoding the amino acid sequences and producing and displaying the encoded peptides.

14. The method of claim 1 further comprising (a) mutating peptides having sequences that are predicted to form secondary structures and (b) identifying redundant sequences in the mutated peptides and removing or deleting redundant sequences to thereby leave a plurality of peptides comprising non-redundant or normalized amino acid sequences.

15. The method of claim 14 comprising performing iterations of (a) and (b) to thereby produce a non-redundant yet highly diverse set of peptides.

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

17. The method of claim 1 wherein the displayed peptides are displayed as a microarray on one solid surface.

18. The method of claim 1 further comprising providing the peptide library comprising the displayed peptides.

19. The method of claim 1 wherein the displayed peptides are displayed on a plurality of solid surfaces.

20. 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.

21. 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.

22. The process of claim 20 further comprising isolating the identified peptide.

23. The process of claim 22 further comprising subjecting the isolated peptide to mutation or an affinity maturation.

24. The process of claim 20 further comprising identifying a mimetic of the structure formed by a peptide.

25. 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.

26. The process of claim 25 further comprising identifying one or more chemical compounds having the secondary structure and/or activity of the peptide.

27. A method for producing a library of independent protein secondary structures having low structural redundancy, said method consisting essentially of the following sequential steps:

(i) executing a computer program to thereby obtain a plurality of amino acid sequences of different proteins from bioinformatic data source(s) wherein the sequences are predicted to form secondary structures and/or assemblies of secondary structures in their native contexts;

(ii) executing a computer program to simultaneously select from (i) a plurality of amino acid sequences of the different proteins, wherein the simultaneously-selected sequences each consist of a single segment of a protein corresponding to a hydrophobic folding unit is predicted to form a secondary structures independent of any other part of the protein from which it is derived when isolated from those other parts of the protein;

(iii) executing a computer program to thereby identify sequences in the plurality at (ii) that are predicted to form redundant secondary structures and removing or deleting sufficient sequences predicted to form the redundant secondary structures to thereby select a plurality of amino acid sequences predicted to form non-redundant secondary structures independent of any other part of the proteins from which they are derived when isolated from those other parts of the proteins, wherein no more than five of said sequences are predicted to form the same secondary structure; and

(iv) chemically-synthesizing a plurality of peptides that each consist essentially of a sequence selected at (iii) that is predicted to form a non-redundant secondary structure independent of any other part of the protein from which it is derived when isolated from those other parts of the protein; and

(v) displaying the plurality of peptides chemically-synthesized at (iv) such that the displayed peptides form secondary structures independent of other parts of the proteins, thereby producing a library of independent protein secondary structures having low structural redundancy.

28. The method according to claim 27 wherein the displayed peptides comprise related secondary structures that differ in their ability to fold autonomously.

29. The method according to claim 27 wherein the displayed peptides comprise related secondary structures that differ in their ligand-binding affinities and/or association/dissociation constants for a ligand.

30. The method according to claim 27 wherein the displayed peptides comprise related secondary structures that differ in their chemical modifications.

31. A method for producing a library of independent protein secondary structures having low structural redundancy, said method consisting essentially of the following sequential steps:

(i) executing a computer program to thereby obtain a plurality of amino acid sequences of different proteins from bioinformatic data source(s) wherein the sequences are predicted to form secondary structures and/or assemblies of secondary structures in their native contexts;

(ii) executing a computer program to simultaneously select from (i) a plurality of amino acid sequences of the different proteins, wherein the simultaneously-selected sequences each consist of a single segment of a protein corresponding to a hydrophobic folding unit is predicted to form a secondary structures independent of any other part of the protein from which it is derived when isolated from those other parts of the protein;

(iii) executing a computer program to identify sequences in the plurality at (ii) which are redundant and removing or deleting the redundant sequences to thereby select for a non-redundant plurality of sequences;

(iv) chemically-synthesizing a plurality of peptides that each consist essentially of a sequence selected at (iii) which is non-redundant and that is predicted to form a secondary structure independent of any other part of the protein from which it is derived when isolated from those other parts of the protein; and

(v) displaying the plurality of peptides chemically-synthesized at (iv) such that the displayed peptides form secondary structures independent of their native contexts, thereby producing a library of independent protein secondary structures having low structural redundancy.

32. A method for producing a library of independent protein secondary structures, said method comprising:

(i) executing a computer program to thereby identify a plurality of amino acid sequences of different proteins from bioinformatic data source(s) wherein the sequences are capable of folding independently from other parts of the proteins in which they are contained in their native contexts;

(ii) executing a computer program to simultaneously select from (i) a plurality of amino acid sequences of different proteins that arc each consist of a single segment of a protein corresponding to a hydrophobic folding unit capable of folding independently from other parts of the proteins from which they are derived when isolated from those other parts of the proteins;

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

(iv) executing a computer program to identify redundant sequences in the sub-set of sequences selected at (iii) and removing or deleting redundant sequences to thereby select for a non-redundant plurality of amino acid sequences;

(v) chemically-synthesizing a plurality of peptides that each consist essentially of a sequence selected at (iv) which is non-redundant and capable of folding independently of any other part of the protein from which it is derived when isolated from other parts of the protein; and

;

(vi) displaying the plurality of peptides chemically-synthesized at (v) such that the displayed peptides form secondary structures and/or folds independent of other parts of the proteins, thereby producing a library of independent protein secondary structures.

33. A method for producing a library of independent protein secondary structures, said method comprising:

(i) executing a computer program to thereby identify a plurality of amino acid sequences of different proteins from bioinformatic data source(s) wherein the sequences are capable of folding independently from other parts of the proteins in which they are contained in their native contexts;

(ii) executing a computer program to simultaneously select from (i) a plurality of amino acid sequences of the different proteins that each consist of a single segment of a protein corresponding to a hydrophobic folding unit and are each capable of folding independently from other parts of the proteins from which they are derived when isolated from those other parts of the proteins;

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

(iv) executing a computer program to identify redundant sequences in the sub-set of sequences selected at (iii) and removing or deleting redundant sequences to thereby select for a non-redundant plurality of amino acid sequences;

(v) identifying related sequences to the non-redundant plurality of amino acid sequences at (iv) and adding those related sequences to the non-redundant plurality of amino acid sequences to thereby produce a diverse pool of amino acid sequences;

(vi) chemically-synthesizing a plurality of peptides that each consist essentially of a sequence selected from the diverse pool of amino acid sequences produced at (v); and

(vii) displaying the plurality of peptides chemically-synthesized at (vi) such that the displayed peptides form secondary structures and/or folds independent of any other parts of the protein from which they are derived when isolated from those other parts of the proteins their native contexts, thereby producing a library of independent protein secondary structures.