# US 7,745,393 B2 · Minimalist bZIP proteins and uses thereof

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- Number: US 7,745,393 B2
- Document ID: US7745393B2
- Status: Granted
- Kind: B2
- Application number: 11/592,186
- Filed: 2006-11-03
- Published: 2007-06-21
- Granted: 2010-06-29
- Earliest claimed: 2005-11-03
- Inventors: Jumi Shin
- Web page: https://patentgrep.com/patent/US7745393B2

## CPC

- C07K 14/4702: {Regulators; Modulating activity}
- 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)

## Abstract

The present invention provides minimalist bZIP proteins having a basic region derived from bHLH proteins fused to a leucine zipper dimerization domain derived from bZIP proteins and methods and uses thereof in the treatment of cancer. The present invention also provides pharmaceutical compositions for treating cancer.

## Claims

### Claim 1

A minimalist bZIP protein consisting of:
a) a basic region of a basic helix-loop-helix protein (bHLH);
b) a hinge region; and
c) a leucine zipper domain of a bZIP protein,
wherein the minimalist bZIP protein binds a target DNA sequence.

### Claim 2 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the hinge region comprises 0-50 amino acids.

### Claim 3 (depends on 2)

The minimalist bZIP protein of claim 2, wherein the hinge region comprises 3 or 4 amino acids.

### Claim 4 (depends on 3)

The minimalist bZIP protein of claim 3, wherein the hinge region comprises the sequence RIR or GIR.

### Claim 5 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the hinge region further comprises an additional 1, 2, or 3 amino acids derived from the C-terminal end of helix 1 of the bHLH protein between the basic region and the hinge region.

### Claim 6 (depends on 1)

The minimalist bZIP protein of claim 1, comprising 30 to 100 amino acids.

### Claim 7 (depends on 6)

The minimalist bZIP protein of claim 6, comprising 40 to 60 amino acids.

### Claim 8 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the bZIP protein is selected from the group consisting of C/EBP, Jun, Fos, GCN4, and CREB.

### Claim 9 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the bHLH protein is selected from the group consisting of a bHLH subvariant, a bHLHZ subvariant, and a bHLH/PAS subvariant.

### Claim 10 (depends on 9)

The minimalist bZIP protein of claim 9, wherein the bHLH subvariant is selected from the group consisting of MyoD, Myc, E2A, E47, E12, TALL, Id proteins, GL3, EGL3, TFEB, PIF1, PILE, ATH, NGN, and HAND1.

### Claim 11 (depends on 9)

The minimalist bZIP protein of claim 9, wherein the bHLHZ subvariant is selected from the group consisting of Mad, Mxi, Max, Myc, Spz1, USF, Mash, BMP, TFE3, and AP4.

### Claim 12 (depends on 9)

The minimalist bZIP protein of claim 9, wherein the bHLH/PAS subvariant is selected from the group consisting of AhR, ARNT, HIF1α, HIF-2α, HIF-3α, Per, and Sim.

### Claim 13 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the target DNA sequence is an E-box sequence selected from 5′-CAG CTG and 5′-CAC-GTG.

### Claim 14 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the target DNA sequence is an XRE1 sequence 5′-TTGC-GTG.

### Claim 15 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the target DNA sequence is a half-site sequence 5′-T(C/T)GC or 5′-GT(A/G)C.

### Claim 16 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the basic region of the bHLH protein has been mutated to generate an alanine rich sequence.

### Claim 17 (depends on 1)

The minimalist bZIP protein of claim 1, comprising the amino acid sequence as shown in SEQ ID NO: 53.

### Claim 18 (depends on 1)

The minimalist bZIP protein of claim 1 comprising the amino acid sequence as shown in SEQ ID NO: 54.

### Claim 19 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the basic region is from Max, the leucine zipper region is from C/EBP, and the target DNA sequence is an E-box sequence.

### Claim 20 (depends on 19)

The minimalist bZIP protein of claim 19, comprising an amino acid sequence selected from the sequences as shown in SEQ ID NOs: 1-6.

### Claim 21 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the basic region is from Arnt, the leucine zipper region is from C/EBP and the target DNA sequence is an XRE1 target DNA sequence.

### Claim 22 (depends on 21)

The minimalist bZIP protein of claim 21, comprising an amino acid sequence selected from the sequences as shown in SEQ ID NOs: 7-12.

### Claim 23 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the protein is further fused to an activation domain.

### Claim 24 (depends on 23)

The minimalist bZIP protein of claim 23, wherein the activation domain is derived from Gal4, Mad, Myc, and VP16.

### Claim 25 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the protein is further fused to a drug for drug delivery.

### Claim 26 (depends on 1)

The minimalist bZIP protein of claim 1, wherein the protein is further fused to a repressor domain.

### Claim 27 (depends on 26)

The minimalist bZIP protein of claim 26, wherein the repressor domain is Mxi, Id, and HIF-3α.

### Claim 28 (depends on 1)

The minimalist bZIP protein of claim 1 comprising the amino acid sequence as shown in SEQ ID NOs: 14-22, 52-54, 56, or 58-64.

### Claim 29

A minimalist bZIP protein heterodimer comprising a first and second minimalist bZIP protein comprising a leucine zipper region in the first minimalist bZIP protein and a leucine zipper region in the second minimalist bZIP protein capable of forming a heterodimer; wherein the first minimalist bZIP protein and the second minimalist bZIP protein each consist of:
a) a basic region of a basic helix-loop-helix protein (bHLH);
b) a hinge region; and
c) a leucine zipper domain of a bZIP protein.

### Claim 30 (depends on 29)

The minimalist bZIP protein heterodimer of claim 29, wherein the leucine zipper region in the first minimalist bZIP protein is from Jun and the leucine zipper in the second minimalist bZIP protein is from Fos.

### Claim 31 (depends on 1)

A pharmaceutical composition comprising the minimalist bZIP protein of claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.