Prediction of essential binding domains for the endocannabinoid N-arachidonoylethanolamine (AEA) in the brain cannabinoid CB1 receptor

Δ9-tetrahydrocannabinol (Δ9-THC), the main active ingredient of Cannabis sativa (marijuana), interacts with the human brain cannabinoid (CB1) receptor and mimics pharmacological effects of endocannabinoids (eCBs) like N-arachidonylethanolamide (AEA). Due to its flexible nature of AEA structure with more than 15 rotatable bonds, establishing its binding mode to the CB1 receptor is elusive. The aim of the present study was to explore possible binding conformations of AEA within the binding pocket of the CB1 receptor confirmed in the recently available X-ray crystal structures of the CB1 receptor and predict essential AEA binding domains. We performed long time molecular dynamics (MD) simulations of plausible AEA docking poses until its receptor binding interactions became optimally established. Our simulation results revealed that AEA favors to bind to the hydrophobic channel (HC) of the CB1 receptor, suggesting that HC holds essential significance in AEA binding to the CB1 receptor. Our results also suggest that the Helix 2 (H2)/H3 region of the CB1 receptor is an AEA binding subsite privileged over the H7 region.

Introduction 43 ∆ 9 -tetrahydrocannabinol (∆ 9 -THC), the main active ingredient of Cannabis sativa 44 (marijuana), interacts with the brain cannabinoid (CB1) receptor and elicits a wide range of 45 neurological, psychological and biological effects [1]. Continuous marijuana use may increase 46 risks of addiction, chronic pain, mood disorders, and birth defects [2,3].  74 The structure of AEA consists of three moieties, including the polar head moiety, the polyene 75 linker moiety, and the hydrophobic tail moiety.

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Our initial motivation of the present study was due to some intriguing results from recent 78 studies demonstrating that the CB1 allosteric modulators (AMs) such as lipoxin A 4 and ZCZ011 79 enhance selectively the AEA-activated CB1 receptors [18,19,20]. As the first step toward 80 understanding how CB1 AMs allosterically enhance AEA-activated CB1 receptors, we felt 81 imperative to determine the binding conformations of AEA responsible for CB1 receptor 82 activation. In the present study, by using a combination of molecular docking and molecular 83 simulations approaches, we explored many possible binding conformations of AEA within the 84 binding pocket of the CB1 receptor and identified essential AEA binding domains. Our results 85 indicate that the hydrophobic channel interactions are crucial for AEA binding to the CB1 86 receptor. Our results also suggest that the H2/H3 region of the CB1 receptor is an AEA binding 87 subsite privileged possibly over the H7 region. were performed more than one hundred times using the best scoring docking pose from the 115 previous run as the starting pose for the next run. For every run the same grid box was used.

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The best scoring docking poses obtained from the above AutoDock runs were overlaid to 117 AM11542 bound to the CB1 receptor in the X-ray crystal structure [6]. Then, depending upon     respect to the initial coordinates after fitting to the heavy atoms of its head moiety (Fig 1).

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Similarly, the RMSD values of the hydrophobic tail moiety of AEA bound to the above fitted 173 CB1 receptor are calculated with respect to the initial coordinates after fitting to the heavy atoms 174 of the hydrophobic tail moiety (Fig 1).  (Table 1). In docking pose4, the head 202 moiety of AEA occupied the deep hydrophobic channel and the tail moiety bound the H2/H3 203 region ( Fig 2B). Thus, docking pose4 was assigned to be 2d_H2/H3 ("2d" denotes that the head 204 moiety binds the deep hydrophobic channel and "H2/H3" denotes the H2/H3 region where the 205 tail moiety binds). In docking pose5, the head moiety occupied the deep hydrophobic channel 206 and the tail moiety bound the H7 region. Thus, docking pose5 was assigned to be 2d_H7. In 207 docking pose6, the head moiety occupies the hydrophobic channel and the tail moiety points 208 toward the middle of the binding core toward the EC region (i.e., the pocket outer core). Thus, 209 docking pose6 was assigned to be 2_OC ("2" denotes Group 2 and "OC" denotes the outer core 210 region). pose8 (a shift from the binding pocket core region to the hydrophobic channel) (Fig 3).  Table 1.   (Table 1). This can be also seen in the RMSD plots of these 270 docking poses (Fig 3).  (Table 1 and Fig 6A). The hydrophobic tail moiety is well    (Table 1 and Fig 6B). The tail moiety occupies the hydrophobic channel just as in the    would also alter AEA binding affinity.

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It is surprising to see in the present study that the polar head moiety of AEA is also able 368 to stably occupy the hydrophobic channel (as in the equilibrated pose 2d_H2/H3) (Fig 6C). It 369 appears that the stabilization of the polar head moiety through H-bonding is required for its 370 binding to the deep hydrophobic channel.  (Table in S1 Table), some ligand  (Table in S1 Table), suggesting with the deep hydrophobic channel is not favored (Fig 3A). Moreover, the equilibrated pose  The chance of the bioactive conformation being present in AEA is much lower than in 445 AM11542 and CP55940, simply because AEA is structurally far more flexible than AM11542 446 and CP55940. It is difficult for the highly flexible AEA to be locked into the active conformation 447 required for best fitting to the binding pocket. Both the varying polar head moiety and the 448 varying hydrophobic tail of AEA would interfere significantly from achieving the bioactive 449 conformation. Overall, AEA is expected to achieve the active conformation much more difficult 450 than AM11542 and CP55940, possibly contributing to its known partial agonistic activity [1].