New insights into the interaction between mammalian butyrylcholinesterase and amitriptyline: a combined experimental and computational approach

Introduction

Butyrylcholinesterase (BChE; EC 3.1.1.8) belongs to the carboxylic ester hydrolase family of enzymes and, in spite of its almost ubiquitous distribution among tissues and organs, is best known for its abundant presence in plasma (hence its alternative names of ‘plasma cholinesterase’ and ‘pseudocholinesterase’). For many years, the physiological function of BChE remained unknown, since individuals with effectively no BChE activity appear to be in good health. This, however, is not in accord with the continued existence of the enzyme through many millions of years in a relatively large number of vertebrate species. It is now established that BChE promotes detoxification by neutralizing drugs, pesticides, and other reactive chemicals including alkaloids both in plasma and in the brain and facilitates neurotransmission by hydrolyzing acetylcholine both in the central nervous system and at the neuromuscular junction of the somatic nervous system [reviewed in [1]. BChE also hydrolyzes ghrelin, possibly modulating levels of this so-called ‘hunger hormone’ in the peripheral circulation 2]. Perhaps paralleling the aforementioned emerging roles of the enzyme, there is a growing body of evidence demonstrating that reduced BChE activity is related with a number of diseases such as liver cirrhosis [3] and chronic obstructive pulmonary disease [4]. In addition, BChE deficiency, which usually arises from natural variations in the gene encoding BChE, leads to prolonged apnea and paralysis following the administration of local anesthetics (e.g. cocaine) and muscle relaxants (e.g. succinylcholine and mivacurium) [reviewed in [5]. On the other hand, elevated BChE activity is associated with Alzheimer’s disease 6] and type 2 diabetes [7]. Putting together these data and the results of numerous studies on this curious enzyme, BChE inhibition is expected to have wide-ranging effects in health and disease.

Amitriptyline (AMI; also known by the trade name Elavil^® among others) is pharmacologically classed as a tricyclic antidepressant (TCA) and works by boosting the neurotransmitters serotonin and norepinephrine/noradrenaline. Although AMI is at least as efficacious as other TCAs or newer drugs, it is linked to some notable side effects (e.g. sedative effects, dry mouth, constipation, and blurred vision) that may be explained by its anticholinergic activity [8]. Çokuğraş and Tezcan [9] were the first to report that AMI inhibited human serum BChE in a reversible, partially competitive mode. That is, using the substrate analog acetylthiocholine (ATCh), they provided evidence showing that the Michaelis constant (K_m) of BChE for its substrate increased and that the maximum velocity (V_m) of the BChE-catalyzed reaction remained unchanged. A plausible explanation for their findings could lie in the ability of AMI to bind at a subsite other than the catalytic subsite (which was postulated by the same authors to be the choline-binding pocket or the acyl-binding pocket) so as to lower the affinity of the enzyme towards its substrate. Intriguingly, Müller et al. [10] found out that AMI inhibited human serum cholinesterase (which was used interchangeably with BChE in the same report) in a reversible, linear mixed-type mode, where it interfered with ATCh binding (thus increasing the K_m) as well as hampered catalysis (thus decreasing the V_m) in a dose-dependent manner. The presence of two distinct AMI-binding sites, among which one was the catalytic subsite, could possibly provide a rational explanation for the occurrence of the aforementioned mixture of competitive and noncompetitive inhibitory effects. Here, using enzyme kinetic measurements as well as protein–ligand docking and interaction profiling, we attempted to resolve the discrepancy between those two experiments and obtain further information on the structural basis of BChE–AMI interaction.

Materials and methods

Results and discussion

Equine BChE shares 90% sequence identity spread over its entire length (i.e. 574 amino acids) with human BChE, despite the current lack of X-ray crystallographic (or nuclear magnetic resonance spectroscopic) data on its three-dimensional structure. Furthermore, all fourteen critical residues constituting the enzyme’s active-site gorge (D70 and Y332 at the peripheral anionic site; W82, Y128, and F329 in the choline-binding pocket; G116, G117, and A199 in the oxyanion hole; S198, E325, and H438 at the catalytic subsite; and W231, L286, and V288 in the acyl-binding pocket) are absolutely conserved between equine BChE and human BChE. When combined with the relatively low cost and biochemically and biophysically well-characterized nature of the commercially available enzyme, the aforementioned structural features make equine serum BChE a valuable mammalian model enzyme providing a reliable basis for human toxicologic and pharmacologic projections. In this study, our primary aim was to gain an accurate and deep understanding of the mechanistic and structural aspects of mammalian BChE inhibition by AMI in an in vitro setting.

When the percent remaining activity of equine serum BChE as a function of AMI concentration was graphed, AMI was found to inhibit the enzyme in a dose-dependent fashion, with an estimated IC₅₀ value of 10 μM (Figure 1). Within the concentration range tested here, percent remaining activity did not reach zero, even though a gradual decrease in BChE activity was noticeable to the human eye. Given that the therapeutic plasma concentration of AMI in major depression is between 80 and 200 ng mL⁻¹ [20], AMI emerges as a potent inhibitor of mammalian BChE at near-therapeutic doses. And considering that the normal plasma concentration of BChE is around 4.64 μg mL⁻¹ [21], the present enzymatic setting maximizes the clinical relevance of our findings.

Figure 1:

Inhibitory activity of AMI against horse serum BChE.

Dose–response curve for the quantification of the relationship between AMI concentration and the inhibitory effect it produces. IC₅₀: half-maximal inhibitory concentration.

Graphing the kinetics of BChE inhibition by AMI, the Lineweaver–Burk plot of 1/specific activity versus 1/[AMI] yielded a family of straight lines intersecting in the second quadrant (Figure 2A). This overall pattern reflects the unique characteristics of mixed-type inhibition, where the effective K_m increases and the effective V_m decreases upon inhibitor binding. As a means to produce helpful insights into the detailed mechanism of BChE–AMI interaction, the replots of slope versus [AMI] and y-intercept versus [AMI] were graphed on the same set of axes (Figure 2B). The resulting pattern illustrated that α is different from and greater than 1. Statistical analysis of the kinetics of BChE inhibition by AMI revealed that the best fit to the experimental data was obtained with a linear mixed-type inhibition model. The corresponding parameter estimates V_m, K_s, K_i, and α were found to be 1070±28 U mg⁻¹ protein, 0.17±0.02 mM, 2.25±0.66 μM, and 3.26±1.52, respectively. These results are in good agreement with the graphical solutions above, further supporting the correct assignment of the mode of inhibition. Because the value of α is small but greater than 1, the mixed-type model becomes a composite of competitive and noncompetitive inhibitory effects. Consequently, AMI binding in the enzyme’s active-site gorge decreases the affinity of mammalian BChE towards BTCh and also decreases the rate of transformation of bound BTCh. Our findings corroborate the study by Müller et al. [10], in which the researchers reported a K_i value of 1.22 μM and an α value of 2.6 for AMI, as derived from their mixed-type model of BChE inhibition. It should be noted, however, that the same research group directly used human serum in their enzyme assays, unlike the highly-purified formulation of equine serum BChE we used. Human plasma is known to also contain paraoxonase, albumin esterase, and acetylcholinesterase, the former two being at high enough levels to play an important part in ester hydrolysis in this body fluid [22]. In their study, Çokuğraş and Tezcan [9] reported a very large α value – that is to say, 1425. It is a known fact that when the α is very large, inhibitor binding prevents the substrate transformation process and the mixed-type model becomes practically identical to competitive inhibition. Although they used a purified formulation of human serum BChE in their enzyme assays, there is serious conflict between their findings and our findings.

Figure 2:

Kinetics of horse serum BChE inhibition by AMI.

(A) Lineweaver–Burk plot for the preliminary depiction of the pattern of the mode of inhibition. AMI concentrations span the steepest, or near-linear, region of the dose–response curve [○, control (0 μM); ●, 0.25 μM; △, 0.5 μM; ▲, 1 μM; □, 2 μM; ■, 4 μM; ◊, 8 μM]. (B) Slope and y-intercept replots for the preliminary identification of the exact mechanism of inhibition. Ki: inhibition constant; α: degree to which inhibitor binding changes the affinity of the enzyme for its substrate.

In an attempt to predict the AMI-binding site(s) on mammalian BChE, we performed a blind docking simulation. The highest-ranked docking solution (calculated binding energy=−8.50 kcal mol⁻¹) showed that AMI penetrated deep into the active-site gorge of BChE, with its three-ring structure facing the choline-binding subsite and its propylidene side chain, which extends from the central carbocyclic ring, terminating close to the catalytic subsite (Figure 3A). The position of AMI in the active-site gorge of BChE was found to be stabilized through a combination of several noncovalent interactions: (i) one of the two outer benzene rings of the three-ring structure established π–π stacking interactions with W82 in the choline-binding pocket; (ii) the other outer benzene ring and the central carbocyclic (imino) ring formed hydrophobic interactions with W82 and A328, respectively; and the tertiary amino group of the propylidene side chain was involved in a hydrogen-bonding interaction with S198 of the catalytic triad (Figure 3B). The engagement of AMI in various interactions with the choline-binding tryptophan may be the main reason causing the lowered affinity of BChE towards BTCh, while its engagement in a hydrogen-bonding interaction with the catalytic serine may explain the lowered rate of BTCh transformation. Although the unique position of AMI relative to the critical residues involved in the binding/hydrolysis of choline esters is likely to add sufficient structural evidence to the linear mixed-type mode of BChE inhibition, we do not exclude the existence of a second (allosteric) AMI-binding site on the enzyme. This bound AMI, however, could only be visualized in an electron density map of a crystal of the BChE–AMI complex.

Figure 3:

BChE–AMI docking and interaction profiling.

(A) Predicted near-native binding pose for AMI in the active-site gorge of horse serum BChE. AMI is shown in stick representation, with the carbon atoms colored in tan. The active-site gorge of horse serum BChE is shown in surface representation, with the peripheral anionic site colored in yellow, the choline-binding subsite colored in magenta, the oxyanion hole colored in cyan, the catalytic subsite colored in orange, and the acyl-binding subsite colored in green. (B) Favorable noncovalent interactions between horse serum BChE and AMI. AMI and the interacting residues are shown in stick representation, with the same color coding as in Figure 3A. Hydrogen-bonding interaction is shown as blue solid line, π–π stacking interactions are shown as green dashed lines, and hydrophobic interactions are shown as gray dashed lines. Images were rendered with the PyMOL Molecular Graphics System, Version 1.8 (Schrödinger, LLC, Portland, OR, USA).

In view of these results, it is tempting to speculate that with long-term use of AMI in major depressive disorder, acquired BChE deficiency and subsequent peri- and postoperative complications may develop. This phenomenon has previously been illustrated in a case where sertraline-associated acquired BChE deficiency resulted in prolonged apnea after general anesthesia with succinylcholine [23]. On the other hand, the observed BChE inhibitory potency of AMI can possibly be exploited in the rational design of new reversible anticholinesterases. Reversible anticholinesterases, such as donepezil, rivastigmine, and galantamine, have been approved by the U.S. Food and Drug Administration (FDA) for the symptomatic treatment of mild-to-moderate Alzheimer’s disease. They are believed to stimulate cholinergic transmission indirectly by inhibiting the activity of acetylcholinesterase (AChE) and thus delaying the hydrolysis of acetylcholine at receptor sites. Given the increasingly prominent role of BChE in the pathophysiology of Alzheimer’s disease [24], AMI may guide researchers through the structure-based design of more potent and selective BChE (and perhaps AChE) inhibitors that can be tested as anti-Alzheimer’s drugs.

Intriguingly, our findings suggest a cholinergic role for AMI in the body, which is justified by BChE inhibition in vitro. This seems to be in conflict with the general view that TCAs result in anticholinergic side effects, the central ones being sedation (especially with AMI), memory impairment, and confusion. These effects most probably arise from the affinities of TCAs for the muscarinic acetylcholine receptors of the human brain [25]. A tolerance to TCAs normally develops after a few days, and their undesirable side effects then gradually subside in severity. Therefore, it is reasonable to assume that the cholinesterase inhibitory activity of AMI may be partly responsible for the increasing tolerance to AMI over time.

Overall, with its IC₅₀ and K_i values in the low-micromolar range AMI is a relatively potent inhibitor of mammalian BChE. Perhaps more importantly, BChE inhibition appears to be clinically relevant at near-therapeutic doses of AMI. The mechanism of the interaction between BChE and AMI is best described by a linear mixed-type model where both competitive and noncompetitive inhibitory regimes are observed. This alters the affinity of the enzyme for its substrate and also impedes the hydrolysis of the bound substrate. We propose that the resulting mechanistic paradigm arises from the concurrent interaction of AMI with both the choline-binding and catalytic subsites of the enzyme’s active-site gorge. Repeated intoxication and/or long-term use of AMI may pose serious health risks by blocking BChE in the circulatory and nervous systems. But considering that TCAs are well studied, widely prescribed and well tolerated (usually at low doses), AMI may serve as a safe template for the design of new anti-Alzheimer’s drugs with cholinesterase-inhibiting activity.