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Small-Molecule Modulation of Read-Through (SMMRT): Discovery of 2-Phenoxyacetanilides as In Vivo Promoters of Dystrophin Synthesis for the Treatment of Duchenne Muscular Dystrophy. Richard G. Wilde et al.

IP.com Disclosure Number: IPCOM000019287D
Publication Date: 2003-Sep-09
Document File: 22 page(s) / 20M

Publishing Venue

The IP.com Prior Art Database

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Slide 1 of 22

Small-Molecule Modulation of Read-Through (SMMRT): Discovery of 2-Phenoxyacetanilides as In Vivo Promoters of Dystrophin Synthesis for the Treatment of Duchenne Muscular Dystrophy Richard Wilde et al.


Slide 2 of 22

Stop Codons in mRNA

2nd base in codon

 

U

C

A

G

 

U

F

F

L

L

S

S

S

S

Y

Y

STOP

STOP

C

C

STOP

W

U

C

A

G

C

L

L

L

L

P

P

P

P

H

H

Q

Q

R

R

R

R

U

C

A

G

A

I

I

I

M

T

T

T

T

N

N

K

K

S

S

R

R

U

C

A

G

G

V

V

V

V

A

A

A

A

D

D

E

E

G

G

G

G

U

C

A

G

   

U A G

1st base in codon

3rd base in codon


Slide 3 of 22

Translation of a Nonsense-Containing

mRNA Molecule

40S

PREMATURE

STOP CODON

start

AUG

40S

60S

start

AUG

40S

60S

               
 

UGA

UAA

AAAAAAA

CAP

CODING REGION

stop

Poly(A) tail

3’UTR

5’UTR

60S


Slide 4 of 22

SMMRT

 

Small molecule

 

40S

start

AUG

40S

60S

start

AUG

40S

60S

               
 

UGA

start

AUG

40S

60S

UAA

AAAAAAA

CAP

CODING REGION

stop

Poly(A) tail

5’UTR

3’UTR

60S

Small Molecule

Modulation of Read-Through

“Nonsense” suppression

Full-length protein is produced

Normal termination not affected

Gentamicin is known to show this effect


Slide 5 of 22

Premature Termination Signaling as a Mechanism for Many Genetic Diseases

5-30% of all genetic disorders result from nonsense mutations

Premature termination signaling results in truncated

  proteins

Cancer: #2 killer in US

Muscular dystrophy: 1/3500 males

Cystic fibrosis: 1/3000

Over 1600 genes associated with diseases


Slide 6 of 22

Premature Termination Signaling as a Mechanism for Many Genetic Diseases

HEART DISEASE: FAMILIAL HYPERCHOLESTEROLEMIA
DUCHENNE MUSCULAR DYSTROPHY
HEMOPHILIA VON WILLEBRAND DISEASE ß-THALASSEMIA

BLOOD DISORDERS

COLLAGEN DISORDERS
OSTEOGENESIS IMPERFECTA
(BRITTLE BONE DISEASE)
CIRRHOSIS

RETINITIS PIGMENTOSA

 
KIDNEY STONES

CANCERS COLORECTAL CARCINOMAS  

APC
MLH1
MSH2

NEUROFIBROMATOSIS
 
NF1/NF2

RETINOBLASTOMA
 
RB1

LUNG BREAST COLON PANCREATIC NON-HODGKIN’S LYMPHOMA OVARIAN ESOPHAGEAL  

p53
WILM’S TUMOR WT1

CYSTIC FIBROSIS
TAY-SACHS DISEASE ATAXIA-TELANGIECTASIA


Slide 7 of 22

Duchenne Muscular Dystrophy

Dystrophin is necessary for

proper muscle function

Muscular dystrophy results

from mutations in the

dystrophin gene

The mdx mouse harbors a

nonsense mutation in the

dystrophin gene

[This slide contains 1 picture or other non-text object]


Slide 8 of 22

HTS Screening

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

300,000 Compounds

In Vitro

590,000 Compounds

Cell Culture

Luciferase reporter construct containing UGA

codon was stably transfected in 293 human

embryonic kidney cells

Drug used at a single concentration and the

level of expressed luciferase was determined

by light detection

Gentamicin was routinely used as a standard

As a control, the solvent (DMSO) was also

employed in the assay without the presence of

drug

Luciferase reporter mRNA w/ UGA codon used

in an in vitro, rabbit reticulocyte lysate

translation reaction

Compound introduced at single concentration

(30 mM)

Fold increase in light intensity in detection of

the luciferase produced was measured


Slide 9 of 22...