Method for measuring alpha value of muscarinic M1 receptor positive allosteric modulators
The present invention provides a useful and efficient screening method for finding a cholinergic muscarinic M1 receptor positive allosteric modulator (M1PAM) with reduced cholinergic side effects. The present invention also provides a method for treating Alzheimer's disease and the like, a method for reducing cholinergic side effects, and the like which use M1PAM selected by the screening method and having a low α value, or the M1PAM and an acetylcholinesterase inhibitor.
1. A method for screening for a cholinergic muscarinic M1 receptor (M1R) positive allosteric modulator (M1PAM) with reduced cholinergic side effects, comprising the following steps (a) to (c):
(a) contacting M1R or a partial peptide thereof with a test compound, acetylcholine (ACh) and detectable M1 antagonist,
(b) measuring α value of the test compound, wherein the α value is calculated as α AB from numeric formula (ii):
Y
=
[
C
]
K
C
+
α
BC
[
B
]
[
C
]
K
B
K
C
1
+
[
A
]
K
A
+
[
B
]
K
B
+
[
C
]
K
C
+
α
AB
[
A
]
[
B
]
K
A
K
B
+
α
AB
[
B
]
[
C
]
K
B
K
C
;
(
ii
)
wherein Y is the percentage of specific binding;
[A], [B], and [C] are respectively the concentration of ACh, the test compound and detectable M1 antagonist;
K A , K B and Kc respectively show the equilibrium dissociation constant of ACh, the test compound and detectable M1 antagonist; and
α AB and α BC are respectively the cooperativities between the test compound and ACh, and the test compound and detectable M1 antagonist,
(c) repeating steps (a) and (b) to screen one or more test compounds and selecting one or more of the test compounds with a low α value, each as an MIPAM, wherein the low a value is associated with reduced cholinergic side effects, and wherein the low α value is greater than 1 and not more than 500, and wherein the M1PAM with reduced cholinergic side effects has not less than 2-fold safety margin between a minimal cognition effective dose and a minimal cholinergic side effect dose in mammals.
2. The method for screening according to claim 1 , wherein the low a value is greater than 10 and not more than 500.
3. The method for screening according to claim 1 , wherein the cholinergic side effect is diarrhea.
4. A method for screening for a cholinergic muscarinic M1 receptor (M1R) positive allosteric modulator (M1PAM) with reduced cholinergic side effects, comprising the following steps (a) to (c):
(a) contacting M1R or a partial peptide thereof with a test compound and detectable ACh,
(b) measuring α value of the test compound, wherein the α value is calculated from numeric formula (i);
Y
=
[
A
]
K
A
+
α
[
A
]
[
B
]
K
A
K
B
1
+
[
A
]
K
A
+
[
B
]
K
B
+
α
AB
[
A
]
[
B
]
K
A
K
B
;
(
i
)
where Y is the percentage of specific binding;
[A] and [B] are respectively the concentration of detectable ACh and the test compound;
K A and K B are respectively the equilibrium dissociation constant of detectable ACh and the test compound; and
α is the cooperativity between the test compound and detectable ACh,
(c) repeating steps (a) and (b) to screen one or more test compounds and selecting one or more of the test compounds with a low α value, each as an MIPAM, wherein the low α value is associated with reduced cholinergic side effects, and wherein the low α value is greater than 1 and not more than 500, and wherein the M1PAM with reduced cholinergic side effects has not less than 2-fold safety margin between a minimal cognition effective dose and a minimal cholinergic side effect dose in mammals.
5. The method for screening according to claim 4 , wherein the low a value is greater than 10 and not more than 500.
6. The method for screening according to claim 4 , wherein the cholinergic side effect is diarrhea.
7. A method for screening for a cholinergic muscarinic M1 receptor (M1R) positive allosteric modulator (M1PAM) with reduced cholinergic side effects, comprising the following steps (a) to (c):
(a) contacting M1R or a partial peptide thereof with a test compound, ACh, and detectable M1PAM,
(b) measuring α value of the test compound, wherein the α value can be calculated as α AB from numeric formula (iii):
Y
=
[
C
]
K
C
+
α
AC
[
A
]
[
C
]
K
A
K
C
1
+
[
A
]
K
A
+
[
B
]
K
B
+
[
C
]
K
C
+
α
AB
[
A
]
[
B
]
K
A
K
B
+
α
AC
[
A
]
[
C
]
K
A
K
C
;
(
iii
)
wherein Y is the percentage of specific binding;
[A], [B], and [C] are respectively the concentration of ACh, the test compound and detectable M1PAM;
K A , K B and K C respectively show the equilibrium dissociation constant of ACh, the test compound and detectable M1PAM; and
α AB and α AC are respectively the cooperativities between ACh and the test compound, and ACh and detectable M1PAM,
(c) repeating steps (a) and (b) to screen one or more test compounds and selecting one or more of the test compounds with a low α value, each as an MIPAM, wherein the low α value is associated with reduced cholinergic side effects, and wherein the low α value is greater than 1 and not more than 500, and wherein the M1PAM with reduced cholinergic side effects has not less than 2-fold safety margin between a minimal cognition effective dose and a minimal cholinergic side effect dose in mammals.
8. The method for screening according to claim 7 , wherein the low a value is greater than 10 and not more than 500.
9. The method for screening according to claim 7 , wherein the cholinergic side effect is diarrhea.
10. A method for treating a mammal, comprising administering a selected M1PAM of claim 1 and an acetylcholinesterase inhibitor to the mammal.
11. A method for treating a mammal, comprising administering a selected M1PAM of claim 4 and an acetylcholinesterase inhibitor to the mammal.
12. A method for treating a mammal, comprising administering a selected M1PAM of claim 7 and an acetylcholinesterase inhibitor to the mammal.