# US 9,517,252 B2 · p53 activating peptides

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- Number: US 9,517,252 B2
- Document ID: US9517252B2
- Status: Granted
- Kind: B2
- Application number: 14/350,390
- Filed: 2012-09-10
- Published: 2015-12-17
- Granted: 2016-12-13
- Earliest claimed: 2011-09-09
- Assignees: Agency for Science, Technology and Research
- Applicants: Walter Goh; Farid John Ghadessy
- Inventors: Walter Goh; Farid John Ghadessy
- Web page: https://patentgrep.com/patent/US9517252B2

## CPC

- A61K 38/16: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof {(enzyme inhibitors A61K38/005)}
- A61K 38/08: Peptides having 5 to 11 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61K 38/10: Peptides having 12 to 20 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 35/00: Antineoplastic agents
- C07K 5/081: {the side chain containing O or S as heteroatoms, e.g. Cys, Ser}
- C07K 7/06: having 5 to 11 amino acids
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/001: {by chemical synthesis}
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/00: Fusion polypeptide
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention is directed to p53 activating peptides. The present further describes methods for generating these peptides and the use of these peptides.

## Claims

### Claim 1

A peptide capable of restoring the sequence-specific DNA binding or transactivation function of a mutant p53, wherein the peptide:
(a) comprises a sequence selected from the group consisting of:
(SEQ ID NO: 55)
S P T T N H
(SEQ ID NO: 56)
M P H L M A C
(SEQ ID NO: 57)
T A L I D I W E H S V L G K G Y P R M S
(SEQ ID NO: 58)
I L N V L P L L A S R K P
(SEQ ID NO: 59)
VSWSACVLLELCNYFPENPIEEEDWACLG
(SEQ ID NO: 60)
R L Q Q V
(SEQ ID NO: 61)
V R H Q H V G A T I L G W K
(SEQ ID NO: 62)
V R H Q H V G A T I L G W KMGWTDH
(SEQ ID NO: 63)
MS P T T N H
(SEQ ID NO: 64)
MM P H L M A C
(SEQ ID NO: 65)
MT A L I D I W E H S V L G K G Y P R M S
(SEQ ID NO: 66)
MI L N V L P L L A S R K P
(SEQ ID NO: 67)
MVSWSACVLLELCNYFPENPIEEEDWACLG
(SEQ ID NO: 68)
MR L Q Q V
(SEQ ID NO: 69)
MV R H Q H V G A T I L G W K
(SEQ ID NO: 70)
MV R H Q H V G A T I L G W KMGWTDH
MLM
(SEQ ID NO: 71)
TTLIDIWEHSVLGKGYPRMS
(SEQ ID NO: 72)
M TTLIDIWEHSVLGKGYPRMS
(SEQ ID NO: 73)
RAVTDRPVPCPNPRLNLVAN
(SEQ ID NO: 74)
M RAVTDRPVPCPNPRLNLVAN
(b) is a variant of a peptide as defined in (a) above; or
(c) is a derivative of a peptide as defined in (a) or (b) above.

### Claim 2 (depends on 1)

The peptide which is a variant according to claim 1 (b), wherein the variant is a fragment of a peptide as defined in claim 1 (a).

### Claim 3 (depends on 1)

The peptide which is a derivative according to claim 1 (c), wherein the derivative is a peptide which has undergone a post-translational modification.

### Claim 4 (depends on 1)

The peptide according to claim 1 wherein the peptide is fused to a heterologous polypeptide sequence.

### Claim 5 (depends on 1)

The peptide according to claim 1 wherein the peptide is a stably cross-linked peptide (a “stapled” peptide) wherein the cross-linked peptides contain at least two modified amino acids that together form an internal cross-link.

### Claim 6 (depends on 1)

The peptide according to claim 1 wherein the mutant p53 is selected from the group consisting of: p53 G245S mutant, p53 R273H mutant p53 R248Q mutant the p53 R175H mutant.

### Claim 7 (depends on 1)

An isolated nucleic acid molecule encoding a peptide according to claim 1.

### Claim 8 (depends on 7)

A vector comprising an isolated nucleic acid molecule according to claim 7.

### Claim 9 (depends on 1, 7, 8)

A pharmaceutical composition comprising a peptide according to claim 1, an isolated nucleic acid molecule according to claim 7, or a vector according to claim 8, wherein the pharmaceutical composition optionally comprises one or more pharmaceutically acceptable excipients, vehicles or carriers.

### Claim 10 (depends on 9)

The pharmaceutical composition according to claim 9 wherein the pharmaceutical composition comprises a further therapeutic compound, for example an anti-cancer agent.

### Claim 11 (depends on 1, 7, 8)

The peptide according to claim 1, an isolated nucleic acid molecule according to claim 7 or a vector according to claim 8 for use in medicine.

### Claim 12

A method for obtaining a peptide which restores the sequence-specific DNA binding of a mutant p53 (mp53) the method comprising:
a) providing a water-in-oil emulsion comprising a plurality of aqueous microcapsules dispersed in the oil phase, wherein the aqueous phase comprises a library of genes encoding a repertoire of randomized peptides, components necessary to transcribe and translate the peptide genes, and mp53 genes, so that a plurality of aqueous microcapsules each comprise a peptide gene, components necessary to transcribe and translate the gene and a mp53 gene;
b) expressing the peptide genes and mp53 genes to produce their respective gene products within the microcapsules, wherein if the peptide gene encodes a peptide which restores the sequence-specific DNA binding of the mp53 then a mp53/peptide/peptide gene complex forms;
c) breaking the emulsion; and
d) enriching for any p53/peptide/peptide gene complexes.

### Claim 13 (depends on 12)

The method according to claim 12 wherein the peptide genes comprise at least one response element (RE) to which mp53 having restored sequence-specific DNA binding can bind to.

### Claim 14 (depends on 13)

The method according to claim 13 wherein the peptide genes comprise at least one response element (RE) to which mp53 having restored sequence-specific DNA binding can bind to, a randomized reading frame, and regulatory elements which are operably linked to the reading frame to thereby enable its expression.

### Claim 15 (depends on 12)

The method according to claim 12 wherein the peptide genes from the p53/peptide/peptide gene complexes are amplified prior to undergoing one or more further rounds of renewed encapsulation in aqueous microcapsules of a water-in-emulsion, expression of the peptide genes within the aqueous microcapsules and the formation of mp53/peptide/peptide gene complexes, breaking of the emulsion and enrichment for any p53/peptide/peptide gene complexes or amplification.

### Claim 16

A method for obtaining a peptide which increases the sequence-specific DNA binding of a wild-type p53 (p53wt) the method comprising:
a) providing a water-in-oil emulsion comprising a plurality of aqueous microcapsules dispersed in the oil phase, wherein the aqueous phase comprises a library of genes encoding a repertoire of randomized peptides, components necessary to transcribe and translate the peptide genes, and p53wt genes, so that a plurality of aqueous microcapsules each comprise a peptide gene, components necessary to transcribe and translate the gene and a p53wt gene;
b) expressing the peptide genes and p53wt genes to produce their respective gene products within the microcapsules, wherein if the peptide gene encodes a peptide which restores the sequence-specific DNA binding of the p53wt then a p53wt/peptide/peptide gene complex forms;
c) breaking the emulsion; and
d) enriching for any p53/peptide/peptide gene complexes.

### Claim 17 (depends on 16)

The method according to claim 16 wherein the peptide genes comprise at least one response element (RE) to which p53wt having increased sequence-specific DNA binding can bind to.

### Claim 18 (depends on 17)

The method according to claim 17 wherein the peptide genes comprise at least one response element (RE) to which p53wt having increased sequence-specific DNA binding can bind to, a randomized reading frame, and regulatory elements which are operably linked to the reading frame to thereby enable its expression.

### Claim 19 (depends on 16)

The method according to claim 16 wherein the peptide genes from the p53/peptide/peptide gene complexes are amplified prior to undergoing one or more further rounds of renewed encapsulation in aqueous microcapsules of a water-in-emulsion, expression of the peptide genes within the aqueous microcapsules and the formation of p53wt/peptide/peptide gene complexes, breaking of the emulsion and enrichment for any p53/peptide/peptide gene complexes or amplification.

### Claim 20 (depends on 1, 7)

A method to treat cancer or a disorder comprising administering to a subject in need thereof a peptide according to claim 1 or an isolated nucleic acid molecule according to claim 7 wherein the subject has cancer associated with a mutant p53 or a disorder characterized by a low level or a low activity of p53.