MicroED as a powerful tool for structure determination of macrocyclic drug compounds directly from their powder formulations

Abstract

Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as act as molecular chameleons and thereby reach intracellular targets. However, due to their complex structures and inherent flexibility, macrocycles are difficult to study structurally and there are limited structural data available. Herein, we use the cryo-EM method MicroED to determine the novel atomic structures of several macrocycles which have previously resisted structural determination. We show that structures of similar complexity can now be obtained rapidly from nanograms of material, and that different conformations of flexible compounds can be derived from the same experiment. These results will have impact on contemporary drug discovery as well as natural product exploration.

Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED

“…The development of high-throughput MicroED with automation has recently been successfully applied in drug discovery for analysis of small molecule therapeutics, [24, 36, 37] and for the structural determination of large and flexible macrocycles. [15] Herein, high-throughput MicroED is successfully used for polymorph screening from the same experiment, without crystallization assays or different sample preparations. Paritaprevir is known to have chameleonic properties and adopts open conformations in aqueous environment and closed conformations in lipophilic environment.…”

“…Using a SerialEM-based high-throughput microcrystal electron diffraction (MicroED) data collection, we recently solved one of the crystal forms of paritaprevir, hereafter referred to as form α. [15] MicroED is a cryogenic electron microscopy (cryo-EM) method for structure determination from tiny crystals of micron to sub-micron size, bypassing the crystallization assay required for conventional X-ray diffraction. [16][17][18] Recent works has shown the ability of MicroED to solve polymorphic structures of small molecules including glycine by time-resolved in situ crystallization, [19] diketopyrrolopyrroles by drop casting on EM grids, [20] indomethacin by crystallization screening, [21] bis-arylacylhydrazone in the presence or absence of hydrates, [22] and vemurafenib by melt crystallization.…”

“…The MicroED sample preparation and grid screening for paritaprevir was performed according to previously described protocols. [15, 24] Two major crystal forms were identified from the same grid preparation using low-magnification whole-grid atlases. Paritaprevir form α yielded needle-like microcrystals with dimensions of 4–10 µm in lengths and 0.1– 0.5 µm in widths (Figure 1b).…”

“…Initial data collection confirmed that the space group and unit cell dimensions of form α match our recently published MicroED structure of paritaprevir. [15] Paritaprevir form β produced rod-like microcrystals (Figure 1c), typically 2–5 µm in lengths and 0.2–0.4 µm in widths. Using automation, MicroED data were recorded on a Thermo-Fisher Falcon III detector at an electron dose rate of 0.01 e-/Å 2 /s and 1 second exposure per frame as the sample stage was continuously rotated from -30° to +30° at 1° per second.…”

Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED

“…Using a SerialEM-based high-throughput microcrystal electron diffraction (MicroED) data collection, we recently solved one of the crystal forms of paritaprevir, hereafter referred to as form α. [ 19 ] MicroED is a cryogenic electron microscopy (cryo-EM) method for structure determination from tiny crystals of micron to sub-micron size, bypassing the crystallization assay required for conventional X-ray diffraction. [ 20 – 22 ] Recent works have shown the ability of MicroED to solve polymorphic structures of small molecules including glycine by time-resolved in situ crystallization, [ 23 ] diketopyrrolopyrroles by drop casting on EM grids, [ 24 ] indomethacin by crystallization screening, [ 25 ] bis-arylacylhydrazone in the presence or absence of hydrates, [ 26 ] and vemurafenib by melt crystallization.…”