# US 8,771,935 B2 · Screening methods for cognitive enhancers

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- Number: US 8,771,935 B2
- Document ID: US8771935B2
- Status: Granted
- Kind: B2
- Application number: 13/364,504
- Filed: 2012-02-02
- Published: 2012-07-05
- Granted: 2014-07-08
- Earliest claimed: 2002-08-19
- Assignees: Dart NeuroScience (Cayman) Ltd.
- Applicants: Timothy P Tully; Roderick E. M. Scott; Rusiko Bourtchouladze
- Inventors: Timothy P Tully; Roderick E. M. Scott; Rusiko Bourtchouladze
- Web page: https://patentgrep.com/patent/US8771935B2

## CPC

- G01N 33/5041: {involving analysis of members of signalling pathways}
- C12Q 1/66: involving luciferase
- C12Q 1/6888: for detection or identification of organisms
- C12Q 1/6897: involving reporter genes operably linked to promoters
- G01N 33/5023: {on expression patterns}
- G01N 33/5058: {Neurological cells}
- G01N 33/5085: {of invertebrates}
- G01N 33/5088: {of vertebrates}
- G01N 33/6896: {Neurological disorders, e.g. Alzheimer's disease}
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 2600/136: Screening for pharmacological compounds
- C12Q 2600/158: Expression markers
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention provides methods for identifying cognitive enhancers able to enhance CREB pathway function. Cognitive enhancers identified in accordance with the invention can be used in rehabilitating an animal with cognitive dysfunction and for enhancing memory or normal cognitive performance (ability or function) in the animal.

## Claims

### Claim 1

A method comprising:
1) screening a plurality of compounds for potential development as-candidate cognitive enhancer compounds by
2) determining the ability of said compounds to enhance cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) pathway function wherein said screening and determining comprises the steps of:
a) contacting cells of neural origin comprising an indicator gene operably linked to a cAMP response element (CRE) promoter with a test compound and with a suboptimal dose of a CREB function stimulating agent simultaneously or sequentially, wherein said CREB function stimulating agent is forskolin;
b) determining indicator activity in said cells of neural origin which have been contacted with said test compound and with said CREB function stimulating agent, wherein said CREB function stimulating agent is forskolin;
c) determining indicator activity in said cells of neural origin which have been contacted with said CREB function stimulating agent alone, wherein said CREB function stimulating agent is forskolin;
d) determining indicator activity in said cells of neural origin which have been contacted with said test compound alone; and
e) determining indicator activity in said cells of neural origin which have not been contacted with said test compound or said CREB function stimulating agent, wherein said CREB function stimulating agent is forskolin;
f) comparing the indicator activity determined in each of steps b) through e)
g) selecting said test compound if:
i) the indicator activity determined in step b) is significantly increased relative to the indicator activity determined in step c); and
ii) the indicator activity determined in step d) is not significantly different relative to the indicator activity determined in step e);
h) repeating steps a) to g) with a range of different concentrations of said test compound selected in step g); and
i) selecting said test compound if:
i) the indicator activity determined in step b) is significantly increased relative to the indicator activity determined in step c); and
ii) the indicator activity determined in step d) is not significantly different relative to the indicator activity determined in step e) in the range of different concentrations of said test compound, and
3) identifying said test compound as a candidate cognitive enhancer compound if said test compound is selected in steps g) and i).

### Claim 2 (depends on 1)

The method of claim 1 wherein said cells of neural origin are contacted with said test compound prior to contact with said CREB function stimulating agent.

### Claim 3 (depends on 1)

The method of claim 1 wherein said cells of neural origin are human neuroblastoma cells.

### Claim 4 (depends on 1)

The method of claim 1 wherein said indicator gene encodes luciferase.

### Claim 5 (depends on 1)

The method of claim 1 wherein steps a) to g) are repeated with a range of at least four different concentrations of the said test compound selected in step g).

### Claim 6 (depends on 1)

The method of claim 1 wherein said cells of neural origin are contacted with said candidate cognitive enhancer compound prior to contact with said CREB function stimulating agent, wherein said CREB function stimulating agent is forskolin.

### Claim 7 (depends on 1)

The method of claim 1 wherein said cells of neural origin are neurons.

### Claim 8 (depends on 7)

The method of claim 7 wherein said neurons are primary hippocampal cells.

### Claim 9

A method comprising:
1) screening a plurality of compounds for potential development as candidate cognitive enhancer compounds by
2) determining the ability of said compounds to enhance cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) pathway function wherein said screening and determining comprises the steps of:
a) contacting host cells comprising an indicator gene operably linked to a cAMP response element (CRE) promoter with a test compound and with a suboptimal dose of a CREB function stimulating agent simultaneously or sequentially, wherein said CREB function stimulating agent is forskolin;
b) determining indicator activity in said host cells which have been contacted with said test compound and with said CREB function stimulating agent, wherein said CREB function stimulating agent is forskolin;
c) determining indicator activity in said host cells which have been contacted with said CREB function stimulating agent alone, wherein said CREB function stimulating agent is forskolin;
d) determining indicator activity in said host cells which have been contacted with said test compound alone; and
e) determining indicator activity in said host cells which have not been contacted with said test compound or said CREB function stimulating agent, wherein said CREB function stimulating agent is forskolin;
f) comparing the indicator activity determined in each of steps b) through e)
g) selecting said test compound if:
i) the indicator activity determined in step b) is increased >100% relative to the indicator activity determined in step c); and
ii) the indicator activity determined in step d) is not significantly different relative to the indicator activity determined in step e); and
h) repeating steps a) to g) with a range of different concentrations of said test compound selected in step g); and
i) selecting said test compound if:
i) the indicator activity determined in step b) is significantly increased relative to the indicator activity determined in step c); and
ii) the indicator activity determined in step d) is not significantly different relative to the indicator activity determined in step e) in the range of different concentrations of said test compound, and
3) identifying said test compound as a candidate cognitive enhancer compound if said test compound is selected in steps g) and i).

### Claim 10 (depends on 9)

The method of claim 9 wherein said host cells are contacted with said test compound prior to contact with said CREB function stimulating agent.

### Claim 11 (depends on 9)

The method of claim 9 wherein said indicator gene encodes luciferase.

### Claim 12 (depends on 9)

The method of claim 9 wherein steps a) to g) are repeated with a range of at least four different concentrations of the said test compound selected in step g).

### Claim 13 (depends on 9)

The method of claim 9 wherein said host cells are cell of neural origin.