Serial Crystallography for Structure-Based Drug Discovery.
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ABSTRACT:
SUBMITTER: Zhu L
PROVIDER: S-EPMC7572805 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Introduction: X-ray crystallography has made important contributions to modern drug development but its application to many important drug targets has been extremely challenging. The recent emergence of X-ray free electron lasers (XFELs) and advancements in serial femtosecond crystallography (SFX) have offered new opportunities to overcome limitations of traditional crystallography to accelerate the structure-based drug discovery (SBDD) process. Areas covered: In this review, the authors describe the general principles of X-ray generation and the main properties of XFEL beams, outline details of SFX data collection and processing, and summarize the progress in the development of associated instrumentation for sample delivery and X-ray detection. An overview of the SFX applications to various important drug targets such as membrane proteins is also provided. Expert opinion: While SFX has already made clear advancements toward the understanding of the structure and dynamics of several major drug targets, its robust application in SBDD still needs further developments of new high-throughput techniques for sample production, automation of crystal delivery and data collection, as well as for processing and storage of large amounts of data. The expansion of the available XFEL beamtime is a key to the success of SFX in SBDD.
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Project description:Microfluidic strategies to enable the growth and subsequent serial crystallographic analysis of micro-crystals have the potential to facilitate both structural characterization and dynamic structural studies of protein targets that have been resistant to single-crystal strategies. However, adapting microfluidic crystallization platforms for micro-crystallography requires a dramatic decrease in the overall device thickness. We report a robust strategy for the straightforward incorporation of single-layer graphene into ultra-thin microfluidic devices. This architecture allows for a total material thickness of only ∼1 μm, facilitating on-chip X-ray diffraction analysis while creating a sample environment that is stable against significant water loss over several weeks. We demonstrate excellent signal-to-noise in our X-ray diffraction measurements using a 1.5 μs polychromatic X-ray exposure, and validate our approach via on-chip structure determination using hen egg white lysozyme (HEWL) as a model system. Although this work is focused on the use of graphene for protein crystallography, we anticipate that this technology should find utility in a wide range of both X-ray and other lab on a chip applications.
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Project description:Serial crystallography (SX) provides an opportunity to observe the molecular dynamics of macromolecular structures at room temperature via pump-probe studies. The delivery of crystals embedded in a viscous medium via an injector or syringe is widely performed in synchrotrons or X-ray free-electron laser facilities with low repetition rates. Various viscous media have been developed; however, there are cases in which the delivery material undesirably interacts chemically or biologically with specific protein samples, or changes the stability of the injection stream, depending on the crystallization solution. Therefore, continued discovery and characterization of new delivery media is necessary for expanding future SX applications. Here, the preparation and characterization of new polysaccharide (wheat starch (WS) and alginate)-based sample delivery media are introduced for SX. Crystals embedded in a WS or alginate injection medium showed a stable injection stream at a flow rate of < 200 nL/min and low-level X-ray background scattering similar to other hydrogels. Using these media, serial millisecond crystallography (SMX) was performed, and the room temperature crystal structures of glucose isomerase and lysozyme were determined at 1.9-2.0 Å resolutions. WS and alginate will allow an expanded application of sample delivery media in SX experiments.
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Project description:Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.
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Project description:X-ray crystallography plays an important role in structure-based drug design (SBDD), and accurate analysis of crystal structures of target macromolecules and macromolecule-ligand complexes is critical at all stages. However, whereas there has been significant progress in improving methods of structural biology, particularly in X-ray crystallography, corresponding progress in the development of computational methods (such as in silico high-throughput screening) is still on the horizon. Crystal structures can be overinterpreted and thus bias hypotheses and follow-up experiments. As in any experimental science, the models of macromolecular structures derived from X-ray diffraction data have their limitations, which need to be critically evaluated and well understood for structure-based drug discovery.This review describes how the validity, accuracy and precision of a protein or nucleic acid structure determined by X-ray crystallography can be evaluated from three different perspectives: i) the nature of the diffraction experiment; ii) the interpretation of an electron density map; and iii) the interpretation of the structural model in terms of function and mechanism. The strategies to optimally exploit a macromolecular structure are also discussed in the context of 'Big Data' analysis, biochemical experimental design and structure-based drug discovery.Although X-ray crystallography is one of the most detailed 'microscopes' available today for examining macromolecular structures, the authors would like to re-emphasize that such structures are only simplified models of the target macromolecules. The authors also wish to reinforce the idea that a structure should not be thought of as a set of precise coordinates but rather as a framework for generating hypotheses to be explored. Numerous biochemical and biophysical experiments, including new diffraction experiments, can and should be performed to verify or falsify these hypotheses. X-ray crystallography will find its future application in drug discovery by the development of specific tools that would allow realistic interpretation of the outcome coordinates and/or support testing of these hypotheses.
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Project description:The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
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Project description:Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.
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Project description:Structure-based drug design is becoming an essential tool for faster and more cost-efficient lead discovery relative to the traditional method. Genomic, proteomic, and structural studies have provided hundreds of new targets and opportunities for future drug discovery. This situation poses a major problem: the necessity to handle the "big data" generated by combinatorial chemistry. Artificial intelligence (AI) and deep learning play a pivotal role in the analysis and systemization of larger data sets by statistical machine learning methods. Advanced AI-based sophisticated machine learning tools have a significant impact on the drug discovery process including medicinal chemistry. In this review, we focus on the currently available methods and algorithms for structure-based drug design including virtual screening and de novo drug design, with a special emphasis on AI- and deep-learning-based methods used for drug discovery.
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Project description:Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2 mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.
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Project description:Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000-10,000 diffraction patterns, which we collected in 3 to maximally 82?minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.
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