Unraveling the Structure of Meclizine Dihydrochloride with MicroED

Abstract Meclizine (Antivert, Bonine) is a first‐generation H1 antihistamine used in the treatment of motion sickness and vertigo. Despite its wide medical use for over 70 years, its crystal structure and the details of protein‐drug interactions remained unknown. Single‐crystal X‐ray diffraction (SC‐XRD) is previously unsuccessful for meclizine. Today, microcrystal electron diffraction (MicroED) enables the analysis of nano‐ or micro‐sized crystals that are merely a billionth the size needed for SC‐XRD directly from seemingly amorphous powder. In this study, MicroED to determine the 3D crystal structure of meclizine dihydrochloride is used. Two racemic enantiomers (R/S) are found in the unit cell, which is packed as repetitive double layers in the crystal lattice. The packing is made of multiple strong N‐H‐Cl− hydrogen bonding interactions and weak interactions like C‐H‐Cl− and pi‐stacking. Molecular docking reveals the binding mechanism of meclizine to the histamine H1 receptor. A comparison of the docking complexes between histamine H1 receptor and meclizine or levocetirizine (a second‐generation antihistamine) shows the conserved binding sites. This research illustrates the combined use of MicroED and molecular docking in unraveling elusive drug structures and protein‐drug interactions for precision drug design and optimization.

intensity) which resulted in a beam size at approximately 1.4 µm.Typical data collection used a constant rotation rate of ~2° per second over an angular wedge of 80° from -40° to +40°, with 0.5s exposure time per frame.Crystal selected for MicroED data collection were isolated and calibrated to eucentric heights to maintain the crystals inside the beam during the rotation.

MicroED data processing.
The MicroED data was saved in mrc format and converted to smv format using the mrc2smv software (https://cryoem.ucla.edu/microed). 2 The converted frames were indexed and integrated by XDS. 3,4Two selected datasets with the highest resolution were scaled and merged using XSCALE, 4 and intensities were converted to SHELX hkl format using XDSCONV. 4The merged dataset showed 80.7% overall completeness, which can be ab initio solved by SHELXT 5 at a resolution of 0.96 Å (Table S1).The structure was refined by SHELXL 6 in Shelxle 7 as a graphical interference to yield the final MicroED structure (Figure 1 and Table S2).

Molecular Docking.
The ligand structures of 1R/1S were extracted from the refined MicroED structure and saved as mol2 files.The structure of levocetirizine was downloaded from CSD database (CSD entries: KIMDOD) and transformed to mol2 files.The Cl -anions, water molecules, and polar hydrogen atoms from amine in the piperazine ring (pKa ≈ 2.12 and 6.55) 8 or ethoxyacetate group (pKa ≈ 2.9) 9 were removed.Then the ligand structures were imported into AutoDock Tools 1.5.7, 10 where all active torsion bonds were made rotatable.
The Cryo-EM structure of histamine H1 receptor (PDB entry: 7DFL) 11 was retrieved from the Protein Data Bank (https://www.rcsb.org/)after removing the ligand and other protein molecules using Pymol 2.5.5. 12Subsequently, hydrogen atoms and charges were computed and added using AutoDock Tools 1.5.7. 10 A grid box measuring 18.75 Å × 18.75 Å × 18.75 Å with 0.375 Å spacing was positioned along the x-, y-, and z-axes.The grid center was determined based on the experimental ligand (histamine) position at coordinates (131.312,132.360, 158.503), see Figure S3.
The AutoDock Vina 1.1.2was used for molecular docking, 13,14 where all active torsion bonds in ligands were set to be flexible, and the receptor was set as a rigid model.The docked complex with the minimum binding energy was exported and analyzed by the Protein-Ligand Interaction Profiler (PLIP) web tool and Pymol 2.5.5 (Figure 3 and Tables S5-S8). 12,15 S3 Hydrogen-bond geometry in meclizine dihydrochloride 1R/1S (Å, °).

R form D-H H•••A D•••A D-H•••A Type
N1-H

S form D-H H•••A D•••A D-H•••A Type
N1′-H Notes: See details in Figure 2.

Figure S1
Figure S1Crystal appearance and diffraction pattern under the TEM.(A-B) Images of items 1 and 2 under the imaging mode (SA 3400×), respectively.The diffraction beam size was highlighted in dashed blue circles; (C-D) Diffraction pattern of items 1 and 2 under diffraction mode (659 mm), respectively.The integration edge was colored in grey rings.

Figure S2
Figure S2The major conformation changes between the crystal structures of 1R/1S and their molecular docking structures.See notations in Figure1B.

Table S1
MicroED data statistics of two selected items of meclizine dihydrochloride.Table S2MicroED data statistics of meclizine dihydrochloride (merged).
Centroids were determined as the center of aromatic phenyl rings in 1R/1S.bDistanceswere measured between centroids.cRelativeorientations were measured by the angles between planes of two aromatic rings.See details in Figure2.
Notes: a

Table S6
Protein-ligand interactions of histamine H1 receptor and 1R complex predicted by molecular docking (Å, °).Distances were measured between centroids determined as the center of aromatic rings.c Angles were measured by the angles between planes of two aromatic rings. b

Table S7
Protein-ligand interactions of histamine H1 receptor and 1S complex predicted by molecular docking (Å, °).Distances were measured between centroids determined as the center of aromatic rings.c Angles were measured by the angles between planes of two aromatic rings. b
Notes: a