Distinct Conformations of Mirabegron Determined by MicroED

Mirabegron, commonly known as "Myrbetriq", has been widely prescribed as a medicine for overactive bladder syndrome for over a decade. However, the structure of the drug and what conformational changes it may undergo upon binding its receptor remain unknown. In this study, we employed microcrystal electron diffraction (MicroED) to reveal its elusive three-dimensional (3D) structure. We find that the drug adopts two distinct conformational states (conformers) within the asymmetric unit. Analysis of hydrogen bonding and packing demonstrated that the hydrophilic groups were embedded within the crystal lattice, resulting in a hydrophobic surface and low water solubility. Structural comparison revealed the presence of trans- and cis- forms in conformers 1 and 2, respectively. Comparison of the structures of Mirabegron alone with that of the drug bound to its receptor,1 the beta 3 adrenergic receptor (β3AR) suggests that the drug undergoes major conformational change to fit in the receptor agonist binding site. This research highlights the efficacy of MicroED in determining the unknown and polymorphic structures of active pharmaceutical ingredients (APIs) directly from powders.


Grid preparation.
Sample preparation followed procedure as described previously. 1 One carbon-coated copper grid (400-mesh, 3.05 mm O.D., Ted Pella Inc.) was pretreated with glow-discharge plasma at 15 mA on the negative mode using PELCO easiGlow (Ted Pella Inc.) for 60s. Around 1 mg of powdery compounds were carefully weighed by a Mettler Toledo (XPR225DR) analytical balance and mixed with a grid in a 10 mL scintillation vial. After gently shaking the vial, the grid was removed and clipped at room temperature.

MicroED data collection.
The clipped grid was loaded in an aligned Thermo Fisher Talos Arctica Cryo-TEM (200 kV, ~0.0251 Å) at 100 K, equipped with a CetaD CMOS camera (4096 × 4096 pixels) and EPUD (Thermo Fisher) software. 1,2 Screening of size-and thickness-suitable microcrystals was done in the imaging mode (LM 210× and SA 3400×). The MicroED data was collected in the diffraction mode with 741 mm diffraction length, 70 µm C2 aperture, and a 50 µm selected area (SA) aperture in the parallel beam condition (45.2% C2 intensity) which resulted in a beam size at approximately 1.4 µm. Typical data collection used a constant rotation rate of ~1° per second over an angular wedge of 100° or 120° from -50° to +50° or -60° to +60°, respectively, with 1s exposure time per frame. Crystals selected for MicroED data collection were isolated and calibrated to eucentric height to maintain the crystal inside the beam during the rotation.
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. 3,4 Three selected datasets with the highest resolution at 0.9 Å were scaled and merged using XSCALE, 4 and intensities were converted to SHELX hkl format using XDSCONV. 4 The merged dataset showed 99.7% overall completeness, which can be ab initio solved by SHELXT 5 with a resolution of 1.01 Å. The structure was refined by SHELXL 6 in Shelxle 7 as a graphical interference to yield the final MicroED structure (Figure 1, Table S1 in Supporting Information).
Scheme S1 Chemical notations of Mirabegron. Conformer 1 was labeled with atom type and numbers, conformer 2 was labeled with atom type and primed numbers. Conformations along C9-C10 and C9'-C10' were highlighted, showing the trans-and cis-form in conformer 1 and 2, respectively.

Figure S4
Major structural differences observed in conformer 1 and 2. The primary torsion differences were highlighted by red arrows. Selected torsion angles were listed for comparison.

Figure S5
Major structural differences observed in conformer 1 and Cryo-EM structure. 9 The primary torsion differences were highlighted by red arrows. Selected torsion angles were listed for comparison. H atoms were omitted for clarity.

Figure S6
Major structural differences observed in conformer 2 and Cryo-EM structure. 9 The primary torsion differences were highlighted by red arrows. Selected torsion angles were listed for comparison. H atoms were omitted for clarity.

Figure S7
Hydrogen bonding and van der Waals interactions between Mirabegron and the active sites of 3AR (PDB entry: 7DH5). 9 Mirabegron was highlighted in violet, and the contact atoms and the residues involved in hydrogen bonding were labeled. H atoms were omitted for clarity.