Figure 1.
(A) Schematic overview of the α4β2 and α3β4 nAChRs architecture (adapted from 6CNJ, 6PV7, and 6PV8). The α-β localization of the orthosteric binding sites, where (S)-nicotine (6CNJ or 6PV7) and AT-1001 (6PV8) bind, are shown. Table 6. PV7, together with an overview of the loop composition of the binding site, is represented. (B) Binding affinities (Ki, nM) for rat α4β2 nAChR (in red) and for human α3β4 nAChR (in bleu) and α4β2 vs. α3β4 binding selectivity (in black) of some title compounds exhibiting the highest α4β2 affinities. (S)-nicotine and A-84543 are reported for comparison. Asterisk on α3β4 affinity of A-84543 indicates that it was determined at the rat subtype.
Figure 1.
(A) Schematic overview of the α4β2 and α3β4 nAChRs architecture (adapted from 6CNJ, 6PV7, and 6PV8). The α-β localization of the orthosteric binding sites, where (S)-nicotine (6CNJ or 6PV7) and AT-1001 (6PV8) bind, are shown. Table 6. PV7, together with an overview of the loop composition of the binding site, is represented. (B) Binding affinities (Ki, nM) for rat α4β2 nAChR (in red) and for human α3β4 nAChR (in bleu) and α4β2 vs. α3β4 binding selectivity (in black) of some title compounds exhibiting the highest α4β2 affinities. (S)-nicotine and A-84543 are reported for comparison. Asterisk on α3β4 affinity of A-84543 indicates that it was determined at the rat subtype.
Figure 2.
Representative structures of α4β2 and α3β4 ligands from the literature, providing useful information regarding inter-species differences of α4β2 vs. α3β4 selectivity.
Figure 2.
Representative structures of α4β2 and α3β4 ligands from the literature, providing useful information regarding inter-species differences of α4β2 vs. α3β4 selectivity.
Figure 3.
(A) Superimposition of the hα4β2 and hα3β4 binding sites complexed with (S)-nicotine (orange residues, backbone cartoons, and red ligand for hα4β2 and light blue residues, backbone cartoons, and ligand for hα3β4) extracted from the cryo-EMs 6CNJ and 6PV7, respectively. (B) Extracts of alignments of human and rat α4, β2, α3, and β4. Residues within 5 Å from the ligand has been underlined in blue, when identical between the species, in red when different.
Figure 3.
(A) Superimposition of the hα4β2 and hα3β4 binding sites complexed with (S)-nicotine (orange residues, backbone cartoons, and red ligand for hα4β2 and light blue residues, backbone cartoons, and ligand for hα3β4) extracted from the cryo-EMs 6CNJ and 6PV7, respectively. (B) Extracts of alignments of human and rat α4, β2, α3, and β4. Residues within 5 Å from the ligand has been underlined in blue, when identical between the species, in red when different.
Figure 4.
Receptor backbones are represented by orange (hα4β2) or cyan (α3β4) cartoons. (A) Comparison between the proposed binding modes of A-84543 at the hα4β2 (PDB ID: 6CNJ, orange ligand and red residues), hα3β4 (PDB ID: 6PV7, cyan ligand and blue residues), and at the rα3β4 (grey ligand and residue). (B) Comparison between the original cryo-EM binding mode of AT-1001 at the AB interface of hα3β4 (PDB ID: 6PV8, cyan ligand and blue residues) with the docked binding pose at the rα3β4 (grey ligand and residue) and (C) at the DE interface.
Figure 4.
Receptor backbones are represented by orange (hα4β2) or cyan (α3β4) cartoons. (A) Comparison between the proposed binding modes of A-84543 at the hα4β2 (PDB ID: 6CNJ, orange ligand and red residues), hα3β4 (PDB ID: 6PV7, cyan ligand and blue residues), and at the rα3β4 (grey ligand and residue). (B) Comparison between the original cryo-EM binding mode of AT-1001 at the AB interface of hα3β4 (PDB ID: 6PV8, cyan ligand and blue residues) with the docked binding pose at the rα3β4 (grey ligand and residue) and (C) at the DE interface.
Figure 5.
(A) Comparison of the proposed binding mode of the high affinity α4β2 ligand (S)-3 at the hα4β2 (yellow ligand, orange residues and orange backbone cartoon, PDB ID: 6CNJ) and at the hα3β4 (green ligand, light blue residues, and light blue backbone cartoons, PDB ID: 6PV7). (B) Docking of the weak binders (S)-4 (light grey), (S)-5 (faded orange), and (S)-6 (cyan) at the hα4β2 (orange residues and orange backbone cartoon, PDB ID: 6CNJ). Unfavorable steric clashes and hydrogen bonds are depicted as dashed orange and black lines, respectively.
Figure 5.
(A) Comparison of the proposed binding mode of the high affinity α4β2 ligand (S)-3 at the hα4β2 (yellow ligand, orange residues and orange backbone cartoon, PDB ID: 6CNJ) and at the hα3β4 (green ligand, light blue residues, and light blue backbone cartoons, PDB ID: 6PV7). (B) Docking of the weak binders (S)-4 (light grey), (S)-5 (faded orange), and (S)-6 (cyan) at the hα4β2 (orange residues and orange backbone cartoon, PDB ID: 6CNJ). Unfavorable steric clashes and hydrogen bonds are depicted as dashed orange and black lines, respectively.
Figure 6.
Proposed binding modes of (S)-7 (dark grey) at the (A) hα4β2 (PDB ID: 6CNJ) and (B) at the hα3β4 (PDB ID: 6PV7) binding sites, both including and removing the structural water (kinked and extended conformation, respectively). Receptor backbones are represented by orange (hα4β2) and cyan (hα3β4) cartoons. Hydrogen bonds are depicted as black dashed lines.
Figure 6.
Proposed binding modes of (S)-7 (dark grey) at the (A) hα4β2 (PDB ID: 6CNJ) and (B) at the hα3β4 (PDB ID: 6PV7) binding sites, both including and removing the structural water (kinked and extended conformation, respectively). Receptor backbones are represented by orange (hα4β2) and cyan (hα3β4) cartoons. Hydrogen bonds are depicted as black dashed lines.
Figure 7.
Receptor backbone and residues are depicted in yellow (hα4β2) and light blue (hα3β4). Unfavourable steric clashes are represented with orange dashed lines. Proposed binding modes at hα4β2 nAChR (adapted from 6CNJ) of the (A) semirigid non-hydroxylated phenyl ethers (S)-8 (purple), (S,R)-10 (cyan), and (S,S)-10 (faded orange); (B) semirigid hydroxylated phenyl ethers (S)-9 (pink), (S,R)-11 (dark green), and (S,S)-11 (orange); and of (C) semirigid piridyl ethers (S)-12 (light cyan), (S)-13 (faded pink), (S,R)-14 (faded orange), and (S,S)-14 (yellow-green). (D) Comparison of the binding modes of (S,R)-14 at the hα4β2 (ligand faded orange) and at the hα3β4 (ligand faded purple) nAChRs.
Figure 7.
Receptor backbone and residues are depicted in yellow (hα4β2) and light blue (hα3β4). Unfavourable steric clashes are represented with orange dashed lines. Proposed binding modes at hα4β2 nAChR (adapted from 6CNJ) of the (A) semirigid non-hydroxylated phenyl ethers (S)-8 (purple), (S,R)-10 (cyan), and (S,S)-10 (faded orange); (B) semirigid hydroxylated phenyl ethers (S)-9 (pink), (S,R)-11 (dark green), and (S,S)-11 (orange); and of (C) semirigid piridyl ethers (S)-12 (light cyan), (S)-13 (faded pink), (S,R)-14 (faded orange), and (S,S)-14 (yellow-green). (D) Comparison of the binding modes of (S,R)-14 at the hα4β2 (ligand faded orange) and at the hα3β4 (ligand faded purple) nAChRs.
Figure 8.
Proposed binding modes of the selective α4β2 ligand (S)-15 (yellow-green) and of the unselective (S)-16 (yellow) at the (A) hα4β2 nAChR and at the (B) hα3β4 nAChR. Receptor backbones are represented with red (hα4β2) and blue (hα3β4) cartoons, while hα4β2 and hα3β4 residues are shown in orange and light blue, respectively. Hydrogen bonds are depicted with black dashed lines.
Figure 8.
Proposed binding modes of the selective α4β2 ligand (S)-15 (yellow-green) and of the unselective (S)-16 (yellow) at the (A) hα4β2 nAChR and at the (B) hα3β4 nAChR. Receptor backbones are represented with red (hα4β2) and blue (hα3β4) cartoons, while hα4β2 and hα3β4 residues are shown in orange and light blue, respectively. Hydrogen bonds are depicted with black dashed lines.
Figure 9.
Binding poses of the high affinity ligands (S,R)-2 (faded orange), (S,R)-29 (yellow-green), and of the weak binders (S,R)-26 (cyan), (S,R)-31 (grey) at the (A) hα4β2 nAChR (adapted from 6CNJ, yellow residues and backbone cartoons) and at the (B) hα3β4 nAChR (adapted from 6PV7, light blue residues and backbone cartoons). Unfavorable and very unfavorable steric clashes are depicted with orange and red dashed lines, respectively.
Figure 9.
Binding poses of the high affinity ligands (S,R)-2 (faded orange), (S,R)-29 (yellow-green), and of the weak binders (S,R)-26 (cyan), (S,R)-31 (grey) at the (A) hα4β2 nAChR (adapted from 6CNJ, yellow residues and backbone cartoons) and at the (B) hα3β4 nAChR (adapted from 6PV7, light blue residues and backbone cartoons). Unfavorable and very unfavorable steric clashes are depicted with orange and red dashed lines, respectively.
Figure 10.
(A) Binding modes of the pyridodioxane-based selective hα4β2 partial agonist (S,R)-33 (green) and of the weak binders (S,R)-34 (pink), (S,R)-35 (grey), and (S,R)-36 (faded orange) at the hα4β2 binding site (adapted from 6CNJ, yellow residues, and yellow backbone cartoons). (B) Superimposition of (S,R)-33 bound at the hα4β2 binding site (green ligand, yellow residues, and yellow backbone cartoons, adapted from 6CNJ) and bound at hα3β4 binding site (cyan ligand, light blue residues, and backbone cartoons, adapted from 6PV7).
Figure 10.
(A) Binding modes of the pyridodioxane-based selective hα4β2 partial agonist (S,R)-33 (green) and of the weak binders (S,R)-34 (pink), (S,R)-35 (grey), and (S,R)-36 (faded orange) at the hα4β2 binding site (adapted from 6CNJ, yellow residues, and yellow backbone cartoons). (B) Superimposition of (S,R)-33 bound at the hα4β2 binding site (green ligand, yellow residues, and yellow backbone cartoons, adapted from 6CNJ) and bound at hα3β4 binding site (cyan ligand, light blue residues, and backbone cartoons, adapted from 6PV7).
Figure 11.
(A) Similar binding poses of (S,R)-33 (green) and (S,S)-37 (grey) at the hα4β2 nAChR, retraced by (B) (S,S)-38 (dark green) and (S,S)-39 (orange), but not by (S,S)-40 (light blue). (C) Superimposition of the binding modes of (S,R)-2 (faded orange) and (S,R)-43 (pink) at the hα4β2 and (D) hα3β4 nAChRs binding sites.
Figure 11.
(A) Similar binding poses of (S,R)-33 (green) and (S,S)-37 (grey) at the hα4β2 nAChR, retraced by (B) (S,S)-38 (dark green) and (S,S)-39 (orange), but not by (S,S)-40 (light blue). (C) Superimposition of the binding modes of (S,R)-2 (faded orange) and (S,R)-43 (pink) at the hα4β2 and (D) hα3β4 nAChRs binding sites.
Figure 12.
Crucial observations regarding α4β2 vs. α3β4 selectivity.
Figure 12.
Crucial observations regarding α4β2 vs. α3β4 selectivity.
Table 1.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds (
S,
R)-
2, (
S)-
3–
7 reported in the literature.
a Tested at rat cortex using [
3H]-epibatidine, unless otherwise specified.
b Tested at membranes of human α3β4 transfected cells.
c Data from Bavo et al. [
33].
d Data from Bolchi et al. [
32].
e Tested at whole at brain preparation using [
3H]-cytisine; from Elliot et al. [
44].
f Data from Bolchi et al. [
7].
Table 1.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds (
S,
R)-
2, (
S)-
3–
7 reported in the literature.
a Tested at rat cortex using [
3H]-epibatidine, unless otherwise specified.
b Tested at membranes of human α3β4 transfected cells.
c Data from Bavo et al. [
33].
d Data from Bolchi et al. [
32].
e Tested at whole at brain preparation using [
3H]-cytisine; from Elliot et al. [
44].
f Data from Bolchi et al. [
7].
|
---|
Compound | α4β2 Ki (μM) a | α3β4 Ki (μM) b | α3β4 Ki/α4β2 Ki |
---|
(S,R)-2 c | 0.012 | 0.310 | 25.8 |
(S)-3 d | 0.0011 | 0.074 | 67.3 |
(S)-4 e | 0.042 | n.a. | n.a. |
(S)-5 f | 0.600 | 4.5 | 7.5 |
(S)-6 f | 0.0312 | 0.946 | 30.3 |
(S)-7 d | 0.0037 | 0.235 | 63.5 |
Table 2.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
8–
14 reported in Bolchi et al. [
32].
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
Table 2.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
8–
14 reported in Bolchi et al. [
32].
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
|
---|
Compound | α4β2 Ki (μM) a | α3β4 Ki (μM) b | α3β4 Ki/α4β2 Ki |
---|
(S)-8 | 9.4 | 0.749 | 0.08 |
(S)-9 | 0.0189 | 0.271 | 14.3 |
(S,R)-10 | 1.55 | 1.3 | 0.84 |
(S,R)-10 + (S,S)-10 | 4.59 | 1.4 | 0.31 |
(S,R)-11 | 0.011 | 0.257 | 23.4 |
(S,S)-11 | 0.192 | 0.752 | 3.9 |
(S)-12 | 7.28 | 0.794 | 0.11 |
(S)-13 | 0.255 | 2.1 | 8.2 |
(S,R)-14 | 0.027 | 5.0 | 185 |
(S,S)-14 | 0.877 | 5.6 | 6.4 |
Table 3.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
15–
20 reported by Bolchi et al. [
7].
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
Table 3.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
15–
20 reported by Bolchi et al. [
7].
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
|
---|
Compound | α4β2 Ki (μM) a | α3β4 Ki (μM) b | α3β4 Ki/α4β2 Ki |
---|
(S)-15 | 0.0237 | 3.1 | 130.8 |
(S)-16 | 0.0038 | 0.030 | 7.9 |
(S)-17 | 0.528 | 0.200 | 0.4 |
(S)-18 | 0.0142 | 1.200 | 84.5 |
(S)-19 | 0.012 | 0.122 | 10.2 |
(S)-20 | 0.330 | 0.947 | 2.9 |
Table 4.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
2,
21–
32.
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
c Data from Pallavicini et al. [
24].
d Data from Bolchi et al. [
5].
e Data from Bolchi et al. [
31].
f Data from Bavo et al. [
33].
Table 4.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
2,
21–
32.
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
c Data from Pallavicini et al. [
24].
d Data from Bolchi et al. [
5].
e Data from Bolchi et al. [
31].
f Data from Bavo et al. [
33].
|
---|
Compound | α4β2 Ki (μM) a | α3β4 Ki (μM) b | α3β4 Ki/α4β2 Ki |
---|
(R,R)-21 | 43.8 c | - | - |
(R,S)-21 | 12.5 c | - | - |
(S,R)-21 | 0.26 c | 1.2 d | 4.6 |
(S,S)-21 | 0.47 c | 8.2 d | 17.4 |
(S,R)-2 e | 0.012 | 0.310 | 25.8 |
(S,S)-2 e | 0.421 | 0.7 | 1.7 |
(S,R)-22 e | 42 | - | - |
(S,R)-23 e | 51 | - | - |
(S,R)-24 e | 97 | - | - |
(S,R)-25 e | 8.1 | - | - |
(S,R)-26 e | 17 | - | - |
(S,R)-27 e | 9.3 | - | - |
(S,R)-28 e | 35 | - | - |
(S,R)-29 f | 0.022 | 0.019 | 0.9 |
(S,R)-30 f | 14 | 4.5 | 0.3 |
(S,R)-31 f | 147 | 2.2 | 0.02 |
(S,R)-32 f | 6.5 | 1.9 | 0.3 |
Table 5.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
33–
36, from Bolchi et al. [
5].
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
Table 5.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
33–
36, from Bolchi et al. [
5].
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
|
---|
Compound | α4β2 Ki (μM) a | α3β4 Ki (μM) b | α3β4 Ki/α4β2 Ki |
---|
(S,R)-33 | 0.41 | 16.2 | 39.5 |
(S,S)-33 | 30.4 | 22 | 0.7 |
(S,R)-34/(S,S)-34 | 2.5 | 12.3 | 4.9 |
(S,R)-35 | 1.64 | 5.8 | 3.5 |
(S,S)-35 | 3.6 | 8.9 | 2.5 |
(S,R)-36 | 43 | - | - |
(S,S)-36 | 30 | - | - |
Table 6.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
37–
44, from Bavo et al. [
33].
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
Table 6.
Binding affinity data at α4β2 and α3β4 nAChRs and α4β2 vs. α3β4 selectivity ratio of compounds
37–
44, from Bavo et al. [
33].
a Tested at rat cortex using [
3H]-epibatidine.
b Tested at membranes of human α3β4 transfected cells.
|
---|
Compound | α4β2 Ki (μM) a | α3β4 Ki (μM) b | α3β4 Ki/α4β2 Ki |
---|
(S,R)-37 | 7.1 | 3.9 | 0.5 |
(S,S)-37 | 0.131 | 13 | 100 |
(S,R)-38 | 12.2 | 1.1 | 0.1 |
(S,S)-38 | 0.335 | 6.7 | 20.0 |
(S,R)-39 | 0.55 | 3.9 | 7.1 |
(S,S)-39 | 1.7 | >100 | >59 |
(S,R)-40 | 0.64 | 2.7 | 4.2 |
(S,S)-40 | 7.3 | 5.3 | 0.7 |
(S,R)-41 | 86 | 3.5 | 0.04 |
(S,R)-42 | 46 | 15 | 0.3 |
(S,R)-43 | 50 | 1.9 | 0.04 |
(S,R)-44 | 59 | 5.4 | 0.1 |