Identifying the binding site of novel methyllycaconitine (MLA) analogs at α4β2 nicotinic acetylcholine receptors

Abstract

Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the α7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat α7, α4β2, and α3β4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC(50)) within an IC(50) range of 2.3-26.6 μM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the α7 nAChR. At α4β2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of α4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of α4β2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6' and 13' in a state-dependent manner, but the reactive probe only bonded with 13' in the open state. The data suggest that the 13' position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (α4)(3)(β2)(2) homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker.

Keywords: Ligand-gated ion channel (LGIC); homology model; methyllycaconitine (MLA); nicotinic acetylcholine receptor (nAChR); noncompetitive antagonist (NCA); reactive probe; substituted cysteine accessibility method (SCAM).

Figure 1

Methyllycaconitine 1 and simple analogs. Azatricyclic anthranilate ester 2, azatricyclic alcohol 3, azabicyclic anthranilate ester 4, azabicyclic alcohol 5, and azabicyclic mustard 6.

Figure 2

Concentration−response curves for ACh alone (◻) and ACh in the presence of compounds (30 μM) 2 (▲), 3 (●), 4 (◼), and 5 (▼) at (A) α4β2 normalized to 1 mM ACh (B) α3β4 normalized to 1 mM ACh and (C) α7 normalized to 10 mM ACh with a 3 min preincubation period. Data are presented as the mean ± SEM (n = 3 − 15 oocytes). All compounds studied were NCAs (represented by a significant drop in I_max and no significant rightward shift of the ACh EC₅₀) at α4β2 and α3β4 nAChRs albeit compound 2 was previously reported (14) to have mixed competitive and noncompetitive properties as there was both a drop in I_max and shift in EC₅₀ (Table 1). At α7, only compounds 3 and 5 were noncompetitive, while compounds 2 and 4 exhibited competitive rather than noncompetitive effects.

Figure 3

(A) Normalized currents (ACh, 100 μM) versus preincubation times with bicyclic alcohol 5 (30 μM) at α4β2 nAChRs. The maximum inhibition of ACh (100 μM) exhibited by bicyclic alcohol 5 was achieved within 3 min, with no further increase thereafter. (B) Concentration-inhibition curves of bicyclic alcohol 5 in the presence of ACh (100 μM) without (●) and with (○) a 3 min preincubation. The IC₅₀ values for bicyclic alcohol 5 with (11.6 μM; 95% CI = 5.2−25.9) and without (53.2 μM; 95% CI = 18.8−150.6) preincubation were statistically different (Student’s t test; p < 0.05). Data are normalized to ACh (EC₅₀; I_{ACh(100 μM)}) and are presented as the mean ± SEM (n = 3−15 oocytes). (C) Concentration−response curves for ACh alone (◻) and ACh in the presence of bicyclic alcohol 5 (30 μM) without preincubation (●) and with a 3 min preincubation (○). Data are normalized to ACh (1 mM; I_max) in the absence of compound and are presented as mean ± SEM (n = 3−5 oocytes).

Figure 4

Voltage-dependence of bicyclic alcohol 5 inhibition at α4β2 wildtype nAChRs. (A) Current−voltage curves obtained by clamping cells (n = 4) between −90 mV and 0 mV in 10 mV steps, with ACh (100 μM) alone (◻) or ACh (100 μM) in the presence of bicyclic alcohol (30 μM) (●). Data is normalized to current generated by ACh (100 μM) alone held at −90 mV. (B) Plot of ACh (100 μM) current block expressed as the ratio of blocked current (30 μM bicyclic alcohol 5) over ACh control current (I_Drug/I_ACh) versus membrane potentials (n = 4). Bicyclic alcohol 5 (30 μM) exerts a stronger block at more negative potentials.

Figure 5

Effect of MTSEA on ACh-induced currents. Currents recorded from oocytes expressing either wildtype α4, α4T2′C, α4S6′C, α4L9′C, α4 V13′C, or α4L16′C with wildtype β2. (A) Current trace from an oocyte expressing α4 V13′Cβ2. Two pulses of ACh (100 μM) were applied before and after a 5 min incubation with 2.5 mM MTSEA + and 1 mM ACh. (B) Trace from an oocyte expressing the α4 V13′Cβ2 mutant. Two pulses of ACh (100 μM) were applied before and after a 3 min preincubation with bicyclic alcohol 5 (1 mM), followed by a 5 min incubation of 2.5 mM MTSEA + 1 mM ACh in the presence of bicyclic alcohol 5. (C) Percentage irreversible change in ACh (1 mM) induced currents in three different conformational states of the channel. Open Channel: 2.5 mM MTSEA + 1 mM ACh incubated for 5 min. Closed Channel: 2.5 mM MTSEA incubated in the absence of ACh for 5 min. Desensitized Channel: 1 mM ACh incubated for 1 min, then 1 mM ACh + 2.5 mM MTSEA incubated for 5 min. All mutants were accessible to sulfyhydryl modification in the open and desensitized channel states. Only α4S6′C was not accessible to MTSEA in the closed channel state and possibly the desensitized state. Data are mean ± SEM (n = 3 − 8 oocytes from at least two batches). Statistical significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001 and compares the effects of MTSEA on the cysteine mutant compared to wildtype. (D) Effects of competing bicyclic alcohol 5 before and after MTSEA on the same mutant. Open Channel: 1 mM bicyclic alcohol 5 was preincubated for 3 min and then treated with 1 mM ACh + 2.5 mM MTSEA for 5 min. Closed Channel: 1 mM bicyclic alcohol 5 incubated for 3 min then competed with 2.5 mM MTSEA in the absence of ACh. Desensitized Channel: 1 mM ACh + 1 mM bicyclic alcohol 5 preincubated for 3 min and then treated with 2.5 mM MTSEA. Data are mean ± SEM of N > 3 oocytes from at least two different batches. Statistical significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001 and compares differences between the effects of MTSEA in the presence and absence of bicyclic alcohol 5 on cysteine mutants.

Figure 6

Synthesis of azabicyclic mustard 6: (a) N,N-bis(ethoxymethyl)benzylamine, CH₃SiCl₃, CH₃CN, 87%; (b) CH₃PPh₃Br, KO^tBu, THF, 58%; (c) α-chloroethyl chloroformate, (CH₂Cl)₂, then CH₃OH, 73%; (d) NaBH₄, chloroacetic acid, PhCH₃, 32%; (e) diisobutylaluminium hydride, CH₂Cl₂, 78%.

Figure 7

(A) Trace showing the effect of ACh (100 μM; duration indicated by black bar) and ACh in the presence of bicyclic alcohol 5 (30 μM; duration indicated by hatched bar) and bicyclic mustard 6 (30 μM; duration indicated by white bar). Both bicyclic alcohol 5 and bicyclic mustard 6 were preincubated for 3 min before coapplying with ACh. (B) Concentration-inhibition curve for bicyclic mustard 6 (○) in the presence of ACh (100 μM) at α4β2 nAChR. The IC₅₀ value was 10.9 μM (95% CI: 2.40−49.8) and was not statistically different (Student’s t test; p > 0.05) to bicyclic alcohol 5 at α4β2 nAChR under the same conditions (Figure 3). (C) Concentration response curves for ACh alone (◻) and ACh in the presence of bicyclic mustard 6 (30 μM) with a 3 min preincubation (Δ) at α4β2 nAChRs. Bicyclic mustard 6 was a noncompetitive antagonist at α4β2 nAChRs (represented by a significant drop in I_max and no significant rightward shift of the ACh EC₅₀ (Table 1).

Figure 8

Effect of bicyclic mustard 6 on ACh-induced currents evoked from oocytes expressing either wildtype α4, α4T2′C, α4S6′C, α4L9′C, α4 V13′C, or α4L16′C mutants with wildtype β2. (A) Example trace showing bicyclic mustard 6 (100 μM) incubated in the presence of ACh (EC₅₀; 100 μM) for 5 min. Two pulses of ACh (100 μM) were applied before and after incubation. (B) Irreversible change in ACh (100 μM) induced currents in presence of bicyclic mustard 6 (100 μM) for the open and the closed states of α4β2 nAChRs. (C) Irreversible change in ACh (100 μM) induced currents in presence of various bicyclic mustard 6 concentrations ranging from 10 to 100 μM at the α4 V13′Cβ2 mutant. Bicyclic mustard 6 shows a concentration-dependent reactivity at the α4 V13′Cβ2 mutant for a given time. Asterisks indicate statistical significance compared to wildtype and are assigned as follows: *p < 0.05; **p < 0.01; ***p < 0.001. All data presented are mean ± SEM (n = 3−8 oocytes).

Figure 9

Comparison of the channel from the template 2BG9 and the homology model used in this study. (A) An amino acid sequence alignment of the α4 and β2 subunits with those in the 2BG9 structure (T. marmorata α, β, δ, and γ). The position of M2 and the pore-lining residues are highlighted. A pairwise sequence comparison (% identity) of α4 with α or δ subunits, and β2 with β or γ subunits is shown to the right. (B) A surface representation of a single α4 subunit from the homology model, with V13′ highlighted in green. The dark line shows the subunit profile that faces the pore. (C) The same profile is plotted as the average pore diameter for the whole pentamer (solid line, (α4)₃(β2)₂ homology model; dotted line, 2BG9) and was calculated using HOLE (18).

Figure 10

Examples of the two main docked poses generated using flexible ligand docking of bicyclic alcohol 5 into a (α4)₃(β2)₂ homology model of the closed channel. The backbones of the M2 helices are shown as ribbons. The side-chains of residues 6′ and 13′ are highlighted, but all others have been removed for clarity. The left-hand panels show the docked pose as seen looking from the extracellular domain, down through the receptor pore. The right-hand panels are the same poses from the side, but with the closest β2 subunit removed so that the ligand can be more clearly viewed. Bicyclic alcohol 5 is shown in CPK.

Figure 11

Examples of the three main docked poses generated using rigid ligand docking of bicyclic alcohol 5 into a (α4)₃(β2)₂ homology model of the closed channel. M2 helices are shown as ribbons, and apart from the side chains of residues 6′ and 13′, all others have been removed for clarity. The left-hand panels show the docked pose as seen looking from the extracellular domain, down through the receptor pore. The right-hand panels are the same poses viewed from the side. Bicyclic alcohol 5 is shown in CPK.