A molecular analysis of substituted phenylethylamines as potential microtubule targeting agents through in silico methods and in vitro microtubule-polymerization activity

Abstract

Natural phenethylamines are trace amine neurotransmitters associated with dopamine transmission and related illnesses such Parkinson's disease, and addiction. Synthetic phenethylamines can have psychoactive and hallucinogenic effects due to their high affinity with the 5-HT_2A receptor. Evidence indicates phenethylamines can directly alter the microtubule cytoskeleton being structurally similar to the microtubule destabilizing agent colchicine, however little work has been done on this interaction. As microtubules provide neuron structure, intracellular transport, and influence synaptic plasticity the interaction of phenethylamines with microtubules is important for understanding the potential harms, or potential pharmaceutical use of phenethylamines. We investigated 110 phenethylamines and their interaction with microtubules. Here we performed molecular docking of these compounds at the colchicine binding site and ranked them via binding energy. The top 10% of phenethylamines were further screened based on pharmacokinetic and physicochemical properties derived from SwissADME and LightBBB. Based on these properties 25B-NBF, 25C-NBF, and DMBMPP were tested in in vitro microtubule polymerization assays showing that they alter microtubule polymerization dynamics in a dose dependent manner. As these compounds can rapidly cross the blood brain barrier and directly affect cytoskeletal dynamics, they have the potential to modulate cytoskeletal based neural plasticity. Further investigations into these mechanisms are warranted.

Figure 1

Structure of colchicine and the top 10% of phenethylamines determined by Autodock Vina binding energy to colchicin’es binding site on tubulin.

Figure 2

A stick and ball representation using Pymol of docked colchicine, 25B-NBF, and 25C-NBF, DMBMPP and the crystal structure of colchicine. The docked structure of colchicine, 25B-NBF, 25C-NBF and DMBMPP follows this atomic coloring scheme: C: yellow, O: red, N: blue, Cl: green, Br: maroon, FL: light blue. (A) Colchicine (yellow) in a docked pose (left), and the overlay of the crystal pose of colchicine (grey) from PDB 4O2B and the docked colchicine (yellow)(right), RMSD = 0.2593 Å. (B) The docked pose of 25B-NBF (yellow) (left), and the overlay of the crystal pose of colchicine (grey) and the docked 25B-NBF (yellow)(right). (C) The docked pose of 25C-NBF (yellow) (left), and the overlay of the crystal pose of colchicine (grey) and the docked 25C-NBF (yellow) (right). (D) The docked pose of DMBMPP (yellow) (left), and the overlay of the crystal pose of colchicine (grey) and the docked DMBMPP (yellow) (right).

Figure 3

Polymerization rate curve of change in optical density at 355 nm over time of (A) 25B-NBF at 10 µM (green), 50 µM (cyan), 75 µM (blue), 100 µM (purple) compared to control (black), positive control 10 µM paclitaxel (red), and negative control 10 µM colchicine (orange); (B) 25C-NBF at 10 µM (green), 50 µM (cyan), 75 µM (blue), 100 µM (purple) compared to control (black), positive control 10 µM paclitaxel (red), and negative control 10 µM colchicine (orange); (C) DMBMPP at 75 µM (blue), 100 µM (purple) compared to control (black), positive control 10 µM paclitaxel (red), and negative control 10 µM colchicine (orange). Averages plotted from a minimum of three biological replicates. Error bars are standard error of the mean.

Figure 4

Fluorescent imaging of control, 10 µM paclitaxel, 10 µM colchicine, 100 µM 25B-NBF, 100 µM 25C-NBF and 100 µM DMBMPP after in vitro polymerization assay at a magnification of 50.4×.