Synthetic cannabinoid receptor agonists are monoamine oxidase-A selective inhibitors

Abstract

Synthetic cannabinoid receptor agonists (SCRAs) are one of the fastest growing classes of recreational drugs. Despite their growth in use, their vast chemical diversity and rapidly changing landscape of structures make understanding their effects challenging. In particular, the side effects for SCRA use are extremely diverse, but notably include severe outcomes such as cardiac arrest. These side effects appear at odds with the main putative mode of action, as full agonists of cannabinoid receptors. We have hypothesized that SCRAs may act as MAO inhibitors, owing to their structural similarity to known monoamine oxidase inhibitors (MAOI's) as well as matching clinical outcomes (hypertensive crisis) of 'monoaminergic toxicity' for users of MAOIs and some SCRA use. We have studied the potential for SCRA-mediated inhibition of MAO-A and MAO-B via a range of SCRAs used commonly in the UK, as well as structural analogues to prove the atomistic determinants of inhibition. By combining in silico and experimental kinetic studies we demonstrate that SCRAs are MAO-A-specific inhibitors and their affinity can vary significantly between SCRAs, most notably affected by the nature of the SCRA 'head' group. Our data allow us to posit a putative mechanism of inhibition. Crucially our data demonstrate that SCRA activity is not limited to just cannabinoid receptor agonism and that alternative interactions might account for some of the diversity of the observed side effects and that these effects can be SCRA-specific.

Keywords: MAO-A; MAO-B; SCRAs; enzyme inhibition; flexible docking analysis; monoamine oxidase; synthetic cannabinoid receptor agonists.

Fig. 1

Structures of compounds investigated in this study. (A) Schematic of general SCRA architecture and alterations made to create SCRA derivatives studies. (B) SCRA structures; These compounds include synthetic cannabinoid receptor agonists (1–5), five compounds that emulate the core and tail section of SCRAs (6–10), benzylamine 11, and kynuramine 12.

Fig. 2

3D protein interactions of 5F‐PB‐22 (3) with residues in the active site of MAO‐A (left) and MAO‐B (right) from docking studies. Hydrophobic interactions have been represented with dashed grey lines, hydrogen bonds with solid blue lines and pi‐stacking interactions with solid green lines. Structure figures were generated using pymol (The PyMOL Molecular Graphics System, Version 2.4.1, Schrödinger, LLC, New York, NY, USA).

Fig. 3

LEFT: Lowest energy binding pose of kynuramine (12, green) into the complex of AM‐2201 (4) in MAO‐A. RIGHT: Lowest energy binding pose of benzylamine (11, pink) into the complex of AM‐2201 (4) in MAO‐B. Compounds 11 and 12 can be seen on the outer surface of the MAO proteins, indicated by a black circle. AM‐2201 (4) is bound to the active site inside both proteins, indicated by a blue circle. The cofactor, FAD, can also be seen under the outer surface of the protein. Structure figures were generated using pymol (The PyMOL Molecular Graphics System, Version 2.4.1, Schrödinger, LLC).

Fig. 4

(A) Steady‐state kinetics plot of MAO‐B turnover, varying [Benzylamine 11], Conditions, 30 μm MAO‐B, 50 mm HEPES, pH 7.5 + 0.5% Triton X‐100. (B) Steady‐state kinetics plot of MAO‐A turnover, varying [kynuramine 12]. Conditions, 20 μm MAO‐A, 50 mm HEPES, pH 7.5 + 0.5% Triton X‐100 All data were recorded in triplicate and error bars represent the standard error.

Fig. 5

Concentration dependence of MAO‐B inhibition by SCRA compounds. (A) Concentration dependence of SCRA compounds 1–3 versus rate of MAO‐B turnover at 25 °C. Solid lines are the fit of the data to Eqn (2). (B) Concentration dependence of compounds 6–10 versus rate of MAO‐B turnover at 25 °C. Solid lines are the fit of the data to Eqn (2). (C) Resulting IC₅₀ values depicting the inhibition potency of compounds 6–10. Conditions, 30 μm MAO‐B, 1.5 mm BZA, 50 mm HEPES, pH 7.5 + 0.5% Triton X‐100, All data were collected in triplicate and error bars indicate standard error.

Fig. 6

Concentration dependence of MAO‐A inhibition by SCRA compounds. MAO‐A turnover in the presence of three SCRA compounds. (A) Concentration dependence of SCRA compounds 1,3,5 versus rate of MAO‐A turnover at 25 °C. Solid lines are the fit of the data to Eqn (2), dashed lines are fit of corresponding MAO‐B data. (B) Resulting IC₅₀ values depicting the inhibition potency of SCRA compounds 1,3,5, with the corresponding IC₅₀ values for MAO‐B shown in pastel. Conditions, 20 μm MAO‐A, 1 mm KYN, 50 mm HEPES, pH 7.5 + 0.5% Triton X‐100, All data were collected in triplicate and error bars indicate standard error.

Fig. 7

Additional kinetic investigation into the SCRA inhibition of MAO‐B turnover. (A) Comparison of in silico and in vitro data; the experimentally determined IC₅₀ values of eight compounds are plotted against the computationally determined docking score. The overlaid heat map indicates the relationship of the maximum % inhibition with respect to the other parameters. (B) Kinetics study of the mechanism of MAO‐B inhibition by compound 8. A Lineweaver‐Burk plot for MAO‐B inhibition by 8 has been plotted where substrate concentrations of 50–3000 mm BZA were used in conjunction with three inhibitor concentrations. (C) Plot of K _M/V _max versus inhibitor concentration for the determination of the K _i value of compound 8. Conditions, 24 μm MAO‐B, 50 mm HEPES, pH 7.5 + 0.5% Triton X‐100, All data were collected in triplicate and error bars indicate standard error.

Fig. 8

Concentration dependence of MAO‐A inhibition by known inhibitor Moclobemide (Structure known above). Concentration dependence of Moclobemide versus rate of MAO‐A turnover at 25 °C. Solid lines are the fit of the data to Eqn (2). Conditions, 20 μm MAO‐A, 1 mm KYN, 50 mm HEPES, pH 7.5 + 0.5% Triton X‐100, All data were collected in triplicate.