Revolutionizing Macrocycle Structural Determination: High-Resolution Insights via Microcrystal Electron Diffraction (MicroED)

Macrocycles have high degree of structural complexity and flexibility which allow them to adapt their conformations to reach intracellular targets and other “undruggable” sites that are not easily reached using conventional small molecules. Despite these advantages, their structural characterization is still a challenge due to the difficulty to obtain high-quality crystals suitable for x-ray crystallography. Macrocycles often exist in multiple conformations which can lead to variations in bioavailability and efficacy which complicates the drug development process. Therefore, there is a need to develop innovative techniques that can provide high-resolution structural data from smaller and less ordered samples. To this end, new study published in the Journal ACS Chemical Biology and conducted by Dr. Emma Danelius, Guanhong Bu, Lianne Wieske, and led by Professor Tamir Gonen from the University of California Los Angeles, developed a novel approach using microcrystal electron diffraction (MicroED). This cryo-electron microscopy (cryo-EM) technique allows for the determination of atomic structures from nanocrystals which are significantly smaller than those required for X-ray diffraction. Moreover, MicroED can obtain high-resolution structural data from minimal sample quantities and is capable of capturing different conformations of flexible compounds from a single experiment which make the technique an ideal tool for studying the difficult to crystallize macrocycles.

To validate the application of MicroED, the team first investigated the structures of brefeldin A and romidepsin. Brefeldin A is an antiviral macrocyclic lactone which the authors selected due to its well-documented structure in x-ray crystallography databases. They successfully collected diffraction data by grinding the compound between two coverslips and applying it directly to electron microscopy grids. The resulting MicroED structure, resolved at 0.85 Å, showed excellent agreement with previously reported X-ray structures and displayed a similar unit cell dimension with an average root mean square deviation (rmsd) of 0.0434 Å. They also evaluated Romidepsin which is a cyclic depsipeptide used as an anticancer agent provided another proof of concept. The compound is a complex structure of 15-membered disulfide ring was similarly prepared and analyzed using MicroED and found to closely match with the known X-ray data. In that experiment, they obtained detailed hydrogen bonding networks and validated further their MicroED’s capability that it can accurately determine the structures of macrocycles with complex intramolecular interactions. The researchers then turned their attention to pacritinib which is a synthetic macrocyclic anticancer agent known for its dual inhibition of Janus kinase 2 (JAK2) and FMS-like receptor tyrosine kinase-3 (FLT3) and pose a challenge due to its flexibility. The authors’ MicroED analysis revealed two distinct conformations in the asymmetric unit significantly different from each other and from the known target-bound structure and this finding highlighted the molecule’s conformational adaptability which is essential for its multitarget activity. According to the authors, the MicroED structure demonstrated how pacritinib’s carbon chains can fold in different directions relative to its aromatic rings and emphasized the importance to capture multiple conformations to understand the molecule’s full therapeutic potential. The team also analyzed simeprevir which is an important NS3/4A protease inhibitor used to treat hepatitis C to study its structural polymorphism and elucidated the MicroED-derived structure resolved at 0.85 Å with two similar conformations in the asymmetric unit. These conformations were consistent with the open and flat configuration observed in the target-bound state which indicates preorganization of the inhibitor into a bioactive conformation. The study also identified hydrogen bonding interactions extending along the crystallographic axis, crucial for understanding the molecule’s binding and solubility characteristics.  Another molecule they studied was troleandomycin which is an antibiotic derived from oleandomycin that has flexible sugar substituents and so to overcome this challenge, the authors employed a molecular replacement approach using an ensemble of calculated conformations to determine its structure via MicroED. According to the authors, the resultant structure showed the sugars in a planar conformation similar to the target-bound state, but with a slightly different macrocyclic core fold and their new method demonstrated the utility of combining computational models with experimental data to solve complex structures. Additionally, Paritaprevir which is a first-generation HCV NS3/4A protease inhibitor, that has high molecular weight and substantial conformational flexibility was resolved at 0.85 Å using MicroED and revealed a relatively open conformation with significant intramolecular hydrogen bonding with large solvent channels along the crystallographic axis, a feature unique among the studied macrocycles. These channels likely play an important role in the drug’s solubility and bioavailability, and possibly influenced its pharmacokinetic properties.

In conclusion, Professor Tamir Gonen and his team at UCLA developed the MicroED capable in obtaining high-resolution structural data of macrocycles from minimal sample quantities. Moreover, the ability to elucidate multiple conformations of macrocycles from a single experiment is a critical because understanding these conformations is essential for optimizing the binding characteristics and therapeutic efficacy of these molecules which often interact dynamically with their targets. The authors also highlighted the potential application of MicroED in guiding the development of multitarget drugs considering the chameleonic nature of macrocycles. Additionally, MicroED can enable structure-based drug design and optimization strategies for more potent, stable, cell-permeable, and orally available macrocyclic drugs. The new MicroED method can inform researchers to optimize macrocyclic compounds for improved pharmacokinetic and pharmacodynamic properties which can lead to the development of more effective drugs with better bioavailability and reduced side effects.