What can MicroED bring to eutectic structure analysis?

   2022-04-24
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The importance of eutectic structure for rational drug design

In recent years, it has become increasingly difficult to obtain effective natural products, and pharmaceutical companies cannot rely on "chance discovery" for innovative drug development. The R&D mindset needs to change from "chance discovery" to "rational design" to get rid of the inherent impression of high investment, long lead time, and high risk in new drug development. Currently, "Rational Drug Design" has become the conventional thinking and approach for innovative drug development by major pharmaceutical companies and Biotech.

Rational Drug Design (RDD) is a structure-based drug design, in which the interaction between the drug structure and the target in vivo is studied to achieve the desired purpose, such as inhibiting the activity of an enzyme, and promoting the release of a substance, or blocking a channel. This process relies heavily on the understanding of the three-dimensional structure of the target and the drug, and thus structural biology has had a profound impact on drug research. For the design of small molecule drugs, the eutectic structure of the target protein-small molecule ligand (hereafter referred to as "eutectic structure") is the most critical information in the drug design process.

The eutectic structure information not only reveals the binding mode and bioactive conformation of both, but also discovers new binding pockets or variable binding sites, and enriches the rational drug design pathways, such as structure-based drug design (SBDD), fragment-based drug design (FBDD), computer-aided drug design (CADD), AI drug discovery, etc.

Methods for obtaining eutectic structures

The 3D structural information of proteins in structural biology studies has been obtained mainly by X-ray crystallography and NMR methods. Among them, NMR can only be tested for samples in solution with very small molecular weights (about 20 kDa) and has been used infrequently in recent years. Therefore, the vast majority of structures in PCBs are obtained by X-ray crystallography (Figure 1 PCB data statistics results).

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Figure 1. Number of XRD and NMR-resolved structures counted by PCB

In the last two decades, the single-particle technique (CryoEM-SPA) developed based on cryo-transmission electron microscopy has made significant progress, and its structural resolution of protein samples can be performed without crystallization. However, the relatively low resolution of cryoEM-SPA (generally >3Å), which is generally more suitable for large molecular weight proteins (>200KD), and the expensive equipment cost also limit the further development of SPA.

Currently, the latest technique for structural biology structure resolution is MicroED, another technique developed based on cryo-transmission electron microscopy, which can test tiny crystal samples using electron beams and complete structure resolution by analyzing diffraction patterns. MicroED can achieve almost the same high resolution as X-ray crystallography, and the single-crystal sample needed for testing only needs to be a hundred nanometers in size, which greatly reduces the difficulty and cycle time of protein single-crystal culture. Given its potential to play a significant role in drug discovery, Science magazine named MicroED as one of the top 10 technological breakthroughs of 2018.


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Figure 2. MicroED was named one of the top 10 technology breakthroughs of 2018 by Science magazine

This paper focuses on the technical features of MicroED and compares it with conventional X-ray crystallography, and gives case studies to illustrate how MicroED can help researchers in drug design to quickly obtain critical eutectic structure information.

MicroED VS. XRD

One hundred years have passed since the discovery of X-ray crystal diffraction by Lauer in 1912 and the pioneering of X-ray crystallography by Bragg Jr. This discovery has had a great impact on the development of human science, especially microstructure science, and is a milestone. In these 100 years, X-ray crystallography, which consists of three steps: crystal growth, diffraction data collection and processing, and structure analysis, has been the biggest bottleneck, where protein particles need to be cultured in solution to become large enough crystals (about 200 μm or more in size). Even in large scientific installations synchrotron radiation, where samples are tested using high-intensity X-rays, it is necessary to grow the sample to a size of 50 microns or more. It is known that crystals of this size are often difficult to obtain for many proteins.

MicroED utilizes the equipment of cryoelectron microscopy to perform structural resolution by bombarding nanocrystals with high-energy electrons to produce diffraction data. MicroED is a diffraction technique, and because the interaction of crystals with high-energy electrons is much stronger than the interaction with X-rays, the crystal size required for MicroED structural resolution is greatly reduced compared to X-ray crystallography, requiring only 100 nanometer-sized crystals are sufficient for testing.

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Figure 3. Comparison of MicroED and X-ray crystallography techniques

For eutectic structure elucidation, which is critical in drug discovery, MicroEDs require significantly lower crystal sizes and therefore offer many application-level advantages.

1. Increased success rate in structural analysis of difficult to crystallize samples
2. Significantly increase the success rate of obtaining eutectic structures
3. Increased efficiency of R&D iterations
4. reduced protein consumption

The following are specific examples of the technical advantages of MicroED.

Advantage of MicroED application (I): Improve the success rate of structural analysis of difficult to crystallize samples and resolve structures that cannot be resolved by XRD

Case 1: HIV-1 protease and the structure of the agent
During HIV virus maturation, HIV-1 protease cleaves Gag protein to assemble mature HIV virus. Therefore, a potential therapeutic strategy is to block the interaction of Gag with HIV-1 protease. The small-molecule inhibitor bevirimat was designed to inhibit the activity of HIV-1 protease, but the eutectic structure of HIV-1-Gag-bevirimat could not be resolved by X-ray crystallography due to difficulties in obtaining crystal samples of sufficient size. Finally, the structure of the HIV-1-Gag-bevirimat eutectic was resolved by MicroED. The structure revealed electrostatic and hydrophobic interactions between bevirimat and the protein, providing a basis for structure-based drug design and the development of more effective inhibitors.

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Fig 4. Structure of HIV-1-Gag-bevirimat resolved by MicroED technique [1]

Case Study 2: MicroED Analysis of Membrane Protein Structures
Lipidic cubic phases (LCP) technique can help membrane proteins to crystallize, thus greatly promoting the development of membrane protein crystallography. However, the crystals of membrane proteins grown by LCP technique are often microcrystalline, and the size of the crystals does not meet the required size for XRD collection but is very suitable for the MicroED technique. The following figure shows the GPCR protein A2AR resolved by the MicroED technique, which is an important potential drug target and has been shown to have important relationships with tumors, Parkinson's disease, drug addiction, and psychiatric diseases.

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Fig 5. Structure of membrane protein A2AR resolved by MicroED technique [2].


Case 3: MicroED determination of peptide structure
Peptides have important biological functions, and resolving the structures of peptides is important for understanding the physiological mechanisms of peptides, but there are peptide structures that cannot be resolved by X-ray crystallography. For example, tau protein aggregation is associated with a variety of neurological diseases (including Alzheimer's disease). tau protein's VQIINK region drives the aggregation and formation of amyloid fibrils in the brain, so inhibition of this region may prevent disease progression. Researchers tried to grow large crystals of this segment but were only able to obtain microcrystals, so the crystal structure of this segment could not be resolved by X-ray methods. Finally, the 1.1 Å resolution structure of the KVQIINKKLD region was resolved using MicroED technology. Based on the resolved structure, the researchers designed a series of inhibitors and found the inhibitor with a good inhibition effect, as shown in Figure 6.

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Fig 6. Significant effect of inhibitors designed according to VQIINK structure [3].

MicroED application advantage (II): Significantly improve the success rate of obtaining eutectic structures

For the acquisition of eutectic structures, the common approach is to achieve them by soaking. Since structural resolution by X-ray crystallography requires protein crystals of sufficient size, the process of soaking small molecules often leads to the fragmentation of large-sized protein single crystals, and eventually, the crystal quality decreases, and eutectic structures cannot be obtained. In contrast, MicroED requires only 100 nm protein crystals, which greatly reduces the chance of fragmentation of small molecules after immersion and increases the space occupancy of small molecules. Therefore, MicroED can significantly improve the success rate of immersion and eutectic structure resolution.

The researchers compared the differences between MicroED and X-ray crystallography in obtaining small protein molecule eutectic by immersion of I3C small molecules in the four binding pockets of previous proteinase K, for example. The results showed that because MicroED uses nanoscale microcrystals, the diffusion of small molecules into the protein pockets is very efficient and the occupancy of small molecules in all four binding pockets is better than in the X-ray resolved structures, as shown in Figure 7. Therefore, the immersion of small molecules into the tiny crystals used in MicroED technology is significantly better than in the large-size crystals used in X-ray crystallography. This will greatly accelerate the FBDD, CADD, and AI drug development process.

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Fig 7. Comparison of small molecule density occupancy in protein binding pockets for XRD and MicroED [4].

MicroED application advantages (3): Improve the efficiency of R&D iteration

(1) XRD requires perfect crystals of large size. Very often, it is difficult to obtain large crystals or obtain large size crystals but there are many defects in them, which require multiple rounds of attempted optimization to improve the crystal quality. MicroED, on the other hand, requires nanocrystals that are easy to obtain and often have fewer defects and higher quality, thus eliminating the need for a lot of optimization time.

(2) Laboratory MicroED devices vs. scarce synchrotron radiation machine time. MicroED devices can be deployed in the laboratory and tested as soon as the same day after getting the sample. In contrast, synchrotron radiation devices are part of the large national basic science equipment, and the machine time is only available once in weeks or months. Therefore, it is often the case that the samples to be tested are prepared, but it takes a long time to wait for the machine time.

MicroED Application Advantage (IV): Reduced Protein Consumption

When protein structure resolution is performed by traditional XRD, as few as hundreds and as many as thousands of crystallographic screens are required, thus protein consumption is high. For some proteins with low expression, the protein production cost cannot be ignored. In contrast, when using MicroED for structural biology research, the number of crystallization screenings required is significantly reduced due to the advantages of low crystal size requirement, low perfection requirement, and high soaking success rate, thus also reducing the consumption of recombinant proteins.

ReadCrystal: A global leading provider of MicroED technology

MicroED technology is uniquely suited for the analysis of eutectic structures of protein-small molecule ligand complexes, which are the primary focus of life sciences and drug discovery.

Based on its self-developed MicroED-related technologies, software, and algorithms, Rexchip provides top-tier international commercial MicroED structural biology services. At the same time, ReadCrystal also provides structural biology services based on synchrotron XRD and CryoEM-SPA cryo-electron microscopy single particle.


Reference:
[1]. PURDY M D, SHI D, CHRUSTOWICZ J, et al. MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat [J]. Proc Natl Acad Sci U S A, 2018, 115(52): 13258-63.
[2]. MICHAEL W. MARTYNOWYCZ A S, ET AL. MicroED structure of the human adenosine receptor 2 determined from a single nanocrystal in LCP [J]. biorix, 2020,
[3]. SEIDLER P M, BOYER D R, RODRIGUEZ J A, et al. Structure-based inhibitors of tau aggregation [J]. Nature chemistry, 2018, 10(2): 170-6.
[4]. MARTYNOWYCZ M W, GONEN T. Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking [J]. Structure, 2021, 29(1): 88-95 e2.