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Small-Molecule Modulation of Read-Through (SMMRT): Discovery of 2-Phenoxyacetanilides as Promoters of PRotein Expression from RNA with Nonsense Codons. Richard G. Wilde et al.

IP.com Disclosure Number: IPCOM000019282D
Publication Date: 2003-Sep-09
Document File: 2 page(s) / 4M

Publishing Venue

The IP.com Prior Art Database

Abstract

A class of 2-phenoxyacetanilides were discovered by HTS as modulators of mRNA read-through for the treatment of genetic diseases such as DMD. A cell-culture assay (with a luciferase reporter containing a nonsense muation) was used to optimize the SAR of the series. Compound 1 was significantly more potent than gentamicin. Compound 1 was stable in buffer solutions, but showed some degradation in mouse serum. Exposure in mice was much higher if dosed subcutaneously over oral dosing. Compound 1 showed superior efficacy in promotion of dystrophin synthesis in mdx mice compared to gentamicin at one-tenth the delivered concentration.

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Slide 1 of 2

1. Introduction

5. Synthesis of Analogues of 1

8. SAR: Carboxy Isosteres

Between 5 and 30% of all genetic disorders (including cystic fibrosis, Duchenne muscular dystrophy, familial hypercholesterolemia, certain cancers, hemophilia, etc.) are caused by nonsense mutations, i.e. the signaling of premature termination of protein expression. The presence of such mutations results in the truncation of vital proteins. For example, lack of dystrophin in DMD patients causes severe muscle wasting and early death.

W

EC2x (mM)

Mf

Cmf

CO2H

0.40

3.40

30

CO2Na

0.40

3.20

10

H

6.9

1.04

10

CN

§

0.27

30

 

§

0.60

100

CO2CH3

§

0.31

30

CONHCH3

§

0.62

100

C2H5

§

0.84

300

 

§

0.59

10

Key: a XCH2COX, TEA, THF; b phenol, K2CO3, MEK; c LiI, pyridine

2. SMMRT: Small Molecule Modulation of Read Through

6. Cell-Culture In Vitro Assay

A luciferase reporter construct containing a UGA codon was stably transfected in cells. Compounds were introduced at ten concen-trations between 0.03 and 300 mM, and luciferase activity was quantified by luminescence. The resulting curves were analyzed as shown below. Typical sd for the EC2x values was ± 0.5 log units.

start

AUG

40S

60S

start

AUG

40S

60S

start

AUG

40S

60S

Truncated Protein

TREAT

DISEASES

MODULATE

TRANSLATION

MODULATE

mRNA STABILITY

9. SAR: The Amide/Ether Linkage

Full-length Protein

           
 
     
     
 
 
     
 

start

AUG

40S

60S

start

AUG

40S

60S

start

AUG

40S

60S

     
 
           
 

start

AUG

40S

60S

           
 
     
 

start

AUG

40S

60S

Many genetic diseases are potential targets

Max fold (Mf)

Light units (protein production)

3. HTS Screen for SMMRT

2 x DMSO

R3

X

R4

Y

R5

EC2x

Mf

Cmf

H

O

H

O

CH3

0.40

3.40

1

CH3

O

H

O

CH3

§

0.36

30

H

S

H

O

CH3

30

1.14

300

H

O

CH3

O

CH3

68

1.16

300

H

O

H

C=O

H

0.74

2.09

300

H

O

H

NH

H

§

0.59

300

H

O

H

S

H

§

0.36

100

DMSO

Luciferase reporter constructs containing a UGA codon were used in two HTS screens. An in vitro screen utilized rabbit reticulocyte lysate and a cell-based assay utilized 293 human embryonic kidney cells. Compounds were introduced at 30 mM, and the amount of luciferase expression was quantified by light detection. A control was measured for the vehicle (DMSO).

EC2x

Cmf

log C

7. SAR: The Benzoic Acid Ring

10. SAR: Position on the Phenol

R1

R2

EC2x (mM)

Mf

Cmf

2-CO2H

H

§

0.13

1

3-CO2H

H

0.40

3.40

30

4-CO2H

H

47

1.84

300

3-CO2H

6-CH3

210

1.10

300

3-CO2H

4-OH

190

1.47

300

3-CO2H

4-Cl

160

1.17

300

3-CO2H

6-Cl

38

1.23

100

3-CO2H

2,4,5-F3

§

0.44

10

3-CO2H

2-CH3

§

0.90

300

4. Identification of a Lead Compound

The HTS screens on > 300,000 compounds identified seven hits. The first compound to be resynthesized and characterized was com-pound 1 (below). This compound was ca. sixty-fold more potent in the full cell-culture assay (above right) compared to

gentamicin, which is the only

known

SMMRT

agent.

R

EC2x (mM)

Mf

Cmf

2-i-Pr

§

0.60

100

3-i-Pr

5.2

2.23

100

4-i-Pr

0.21

2.94

30

3-CH3

7.9

1.30

30

4-CH3

§

0.71

1

1

§ - Never achieved 2-fold over DMSO

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Slide 2 of 2

13. SAR: Bi...