Discover key drug target structures

Researchers from the Moscow Institute of Physics and Technology have published a study on femtosecond serial crystallography, one of the most promising methods for the badysis of the tertiary structure of proteins. This technique has evolved rapidly over the last decade, opening up new perspectives for the rational design of drugs targeting proteins previously beyond the reach of structural badysis. The article is out in the newspaper Expert Opinion on Drug Discovery.

X-ray crystallography

X-ray crystallography is one of the main ways to reveal the 3D structure of biological macromolecules, such as proteins. He helped determine the structure of many pharmacologically important enzymes and receptors, allowing the design of drugs targeting these proteins.

The method consists of crystallizing a protein and studying it by X-ray diffraction. The protein is first isolated and purified. Then the solvent dries out gradually. As a result, molecules whose structure is being studied form crystals characterized by an internal order. By exposing an X-ray crystal in a special device, researchers get a diffraction pattern. It contains information about the positions of atoms in the crystal. Careful badysis of the pattern reveals the 3D structure of the constituent protein molecules.

Before the advent of this method, new drugs were mainly investigated empirically: either by modifying the structure of molecules affecting the target protein, or by sorting arrays of molecules in chemical libraries. Now that 3D structures of many target proteins are available, researchers can view them on a computer screen and quickly sort through millions of compounds in search of drug candidates. This saves a lot of time and money previously spent on chemical synthesis and "wet" experiments.

X-ray crystallography gives good results for large, stable and homogeneous crystals, that is to say without impurities or structural defects. To better detect a weak diffraction signal, a powerful radiation pulse is needed, but not powerful enough to destroy the crystal. In conventional X-ray crystallography, a protein crystal is rotated in the X-ray beam to produce diffraction patterns for various spatial orientations. This captures a maximum of information about the structure.

Method for difficult targets

Shortly after the appearance of X-ray crystallography, it became clear that not all biological macromolecules can be crystallized. Some proteins are usually dissolved in the internal cellular environment. It is therefore quite easy to put them in solution, evaporate them and get a big regular crystal. But membrane proteins, including many receptors, form crystals that are neither large enough nor sufficiently pure for standard X-ray crystallography. That said, many of these proteins are involved in the development of the disease, which means that their structure is of great interest to pharmacologists.

Less than a decade ago, a solution was found for membrane proteins. This new technique, called femtosecond X-ray crystallography in series, or SFX, is based on X-ray free electron lasers, developed shortly before SFX.

Alexey Mishin, deputy director of the Laboratory of Structural Biology Receptors at MIPT, co-author of the study, explained: "Making it a breakthrough technology is the very high energy density of the 39. Laser pulse.The object is exposed to an inevitably and almost instantly, it falls apart.But before that, some individual quanta of the laser pulse disperse on the sample and end up at the detector. the so-called diffraction principle before destruction for the study of the structure of the original protein ".

X-ray free-electron lasers have proven useful outside of biology: in recent years, physicists and chemists have increasingly used SFX. The first device was made available to experimenters in 2009 and five centers are now open to researchers in the United States, Japan, South Korea, Germany and Switzerland. A new building is under construction in China and the US factory – the first in history – has announced modernization projects.

While new technology has provided researchers with insight into the structure of proteins that were previously untested, she has also proposed new technical and mathematical solutions. Conventional X-ray crystallography involves exposing a crystal to radiation from different angles and collectively badyzing the diffraction patterns obtained. In SFX, the crystal is instantly destroyed by the first interaction with a powerful X-ray pulse. Researchers must therefore repeat the process with many small crystals and badyze the "serial" data thus generated, hence the name of the method.

Another challenge is to select the samples for SFX. In conventional X-ray crystallography, the choice of the largest crystal and the highest quality was the solution. This could be done manually, looking at the available samples. The new procedure requires working with a suspension of many small crystals of varying size and quality. Centrifuges and filters with known pore sizes are used to separate crystals by size.

Ways to place samples in the room also had to be worked out. X-ray free electron lasers have a certain maximum frequency at which they can emit radiation pulses. To reduce expense and time consumption, new crystals must be introduced into the chamber at the same frequency. Until now, two approaches have been developed to do this. Under the first, the crystals enter the chamber in a liquid suspension fed by an injector. The jet coming out of the injector is "squeezed" by a stream of gas to ensure the correct placement of the sample. In other words, when it is crossed, a crystal is precisely in the center of the laser beam. Alternatively, the protein crystals can be spread on an X-ray transparent substrate and automatically introduced into the laser beam before each pulse.

Since its first results in 2011, SFX has revealed more than 200 protein structures. Of these, 51 potentially important targets for pharmacology – membrane receptors, enzymes, viral proteins, etc. – were previously inaccessible to conventional badysis techniques.

The systematic review of technology applied to biology and pharmacology by the MIPT team will undoubtedly help other researchers seeking key drug target structures for developing new drugs.

Reference: Alexey Mishin, et al. A perspective on the use of serial femtosecond crystallography in drug discovery. Expert Opinion on Drug Discovery (2019) DOI: https://doi.org/10.1080/17460441.2019.1626822

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