Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

Применение программного продукта «Яндекс.Сервер» для организации поиска в электронном каталоге библиотеки

The huge amounts of information accumulated by libraries in recent years put before developers a problem of the organization of fast and qualitative search which decision is possible with the use of modern search tools of Web-technology. The author examines one of these tools the software product “Yandex. Server”, allowing to organize optimum search in the electronic library catalog. The software product “Yandex. Server” gives a chance to carry out optimum search taking into account morphology of Russian and English languages, as well as the various logical conditions that provides effective and flexible search in the electronic library catalog.Накопленные библиотеками за последние годы огромные массивы информации ставят перед разработчиками задачу организации быстрого и качественного поиска, решение которой возможно с использованием современных поисковых инструментов веб-технологии. Автор рассматривает один из таких инструментов - программный продукт «Яндекс. Сервер», позволяющий организовать оптимальный поиск в электронном каталоге библиотеки с учетом морфологии русского и английского языков, а также различных логических условий

Development of diffraction analysis methods for serial crystallography

Serial crystallography, initially developed for use at X-ray free-electron lasers, has openednew opportunities to investigate structure and dynamics of biomolecules at physiologicallyrelevant temperatures. It has since spread out to 3 rd generation synchrotron sources whereit allows us to measure protein microcrystals at room temperature, perform time-resolvedexperiments on biological crystals and obtain structures of radiation-sensitive proteins. Lately,extending the method of serial synchrotron crystallography to polychromatic X-ray beams hasbecome of particular interest. Polychromatic beams provide two orders of magnitude higherphoton flux, allowing significantly reduced exposure times compared to synchrotron experi-ments with monochromatic X-rays. This, in turn, allows accessing much shorter timescales intime-resolved diffraction experiments at synchrotrons.Serial crystallography is based on merging data from still diffraction patterns collectedfrom small randomly-oriented crystals only once exposed by X-rays, which differs significantlyfrom conventional crystallography where one crystal is measured in different orientationswhile being rotated in the X-ray beam. Therefore, serial crystallography requires specific dataanalysis techniques capable of assembling a complete three-dimensional dataset of structurefactor moduli from large numbers of individual still diffraction patterns. Analysis of serialcrystallographic data has proven to be a complex problem, and despite the huge progress madein the field in the last decade, there is still a lot of room for improvement.The aim of this dissertation is the development of new approaches to the processing andanalysis of serial crystallographic data. Several experiments at both FELs and synchrotronsare presented to illustrate different analysis techniques. In particular, the major topic ofthe dissertation is extending the existing analysis software to serial crystallography withpolychromatic beams. Following the first proof-of-principle study with the bandwidth of 2.5%of a 15 keV X-ray beam, a full data analysis pipeline for pink-beam serial crystallographyis developed. The pipeline is then applied to three different datasets collected with the fullundulator bandwidth of 5%, which demonstrates its feasibility even for the particularly difficultcases. The advantages of the analysis pipeline include the possibility of automated processing oflarge amounts of data, and analysis of polychromatic diffraction data from small crystals below10 micron in size. This opens up new possibilities for time-resolved studies of irreversiblebiological reactions at sub-nanosecond timescales

XGANDALF – extended gradient descent algorithm for lattice finding

Serial crystallography records still diffraction patterns from single, randomly oriented crystals, then merges data from hundreds or thousands of them to form a complete data set. To process the data, the diffraction patterns must first be indexed, equivalent to determining the orientation of each crystal. A novel automatic indexing algorithm is presented, which in tests usually gives significantly higher indexing rates than alternative programs currently available for this task. The algorithm does not require prior knowledge of the lattice parameters but can make use of that information if provided, and also allows indexing of diffraction patterns generated by several crystals in the beam. Cases with a small number of Bragg spots per pattern appear to particularly benefit from the new approach. The algorithm has been implemented and optimized for fast execution, making it suitable for real-time feedback during serial crystallography experiments. It is implemented in an open-source C++ library and distributed under the LGPLv3 licence. An interface to it has been added to the CrystFEL software suite.German Ministry of Education and Research (BMBF) German Research Foundation (DFG) Gottfried Wilhelm Leibniz Program and Clusters of Excellence `Center for Ultrafast Imaging Advanced Imaging of Matte

pinkIndexer – a universal indexer for pink-beam X-ray and electron diffraction snapshots

A crystallographic indexing algorithm,pinkIndexer, is presented for the analysisof snapshot diffraction patterns. It can be used in a variety of contexts includingmeasurements made with a monochromatic radiation source, a polychromaticsource or with radiation of very short wavelength. As such, the algorithm isparticularly suited to automated data processing for two emerging measurementtechniques for macromolecular structure determination: serial pink-beam X-raycrystallography and serial electron crystallography, which until now lackedreliable programs for analyzing many individual diffraction patterns fromcrystals of uncorrelated orientation. The algorithm requires approximateknowledge of the unit-cell parameters of the crystal, but not the wavelengthsassociated with each Bragg spot. The use ofpinkIndexeris demonstrated byobtaining 1005 lattices from a published pink-beam serial crystallography dataset that had previously yielded 140 indexed lattices. Additionally, in tests onexperimental serial crystallography diffraction data recorded with quasi-monochromatic X-rays and with electrons the algorithm indexed more patternsthan other programs tested

Serial and Macromolecular Crystallography at Beamline P11

P11 at PETRA III (DESY, Hamburg) is a high-throughput instrument for macromolecular crystallography (1). P11 has tuneable photon energy between 5.5 - 28 keV having the Eiger2 X 16M as the stationary detector and the possibility of using a CdTe-detector for higher energies. Beam size from 200 x 200 μm to 4 x 9 μm can be used with a maximum photon flux of 1.3 x 10^13 ph/s at 12 keV energy. The automatic sample changer is based on the unipuck format with a total capacity of 23 pucks having an mount-unmount cycle of approximately 36 s, which brings the beamtime down to ca. 2 min per sample. P11 is a very diverse environment and the experimental hutch can accommodate various non-standard experiments e.g. via the long term proposal (LTP) scheme. For example, serial synchrotron crystallography (SSX) is enabled with sample delivery through various types of solid supports or the TapeDrive setup, which allows time-resolved room temperature experiments by the mix-and-diffuse method (2), and has been developed through the LTP scheme along with the real-time autoprocessing with CrystFEL (3). Furthermore, SSX data collections can be combined with inserting a chopper wheel that produces short X-ray pulses for time-resolved experiments. The SSX experiments are controlled through a graphical user interface, and online data analysis is available for real time evaluation and indexing via OnDA Monitor (4).This year MXCuBE will be employed as the default control software with the integration to ISPyB for tracking shipments, communicating the sample details to MXCuBE, as well as acting as a data archive. The establishment of parallel autoprocessing pipelines in addition to the currently used, XDSAPP-based (5) pipeline, and the implementation of strategy calculation including dose estimation are also planned. Furthermore, these software developments are synchronising P11 with the EMBL PETRA III beamlines for the future foundation of a uniform structural biology village at PETRA IV.[1] Burkhardt A., et al., Status of the crystallography beamlines at PETRA III. Eur. Phys. J. Plus 131, 56 (2016)[2] Beyerlein K. R., et al., Mix-and-diffuse serial synchrotron crystallography. IUCrJ 4, 769-777 (2017)[3] White T. A., et al., Recent developments in CrystFEL. J. Appl. Cryst. 49, 680-689 (2016)[4] Mariani V., et al., OnDA: online data analysis and feedback for serial X-ray imaging. J. Appl. Cryst. 49, 1073-1080 (2016)[5] Sparta KM, et al., XDSAPP2.0. J. Appl. Cryst. 49, 1085-1092 (2016

Pink-beam serial femtosecond crystallography for accurate structure-factor determination at an X-ray free-electron laser

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables essentially radiation-damage-free macromolecular structure determination using microcrystals that are too small for synchrotron studies. However, SFX experiments often require large amounts of sample in order to collect highly redundant data where some of the many stochastic errors can be averaged out to determine accurate structure-factor amplitudes. In this work, the capability of the Swiss X-ray free-electron laser (SwissFEL) was used to generate large-bandwidth X-ray pulses [$Δλ/λ$ = 2.2% full width at half-maximum (FWHM)], which were applied in SFX with the aim of improving the partiality of Bragg spots and thus decreasing sample consumption while maintaining the data quality. Sensitive data-quality indicators such as anomalous signal from native thaumatin micro-crystals and de novo phasing results were used to quantify the benefits of using pink X-ray pulses to obtain accurate structure-factor amplitudes. Compared with data measured using the same setup but using X-ray pulses with typical quasi-monochromatic XFEL bandwidth (Δλ/λ = 0.17% FWHM), up to fourfold reduction in the number of indexed diffraction patterns required to obtain similar data quality was achieved. This novel approach, pink-beam SFX, facilitates the yet underutilized de novo structure determination of challenging proteins at XFELs, thereby opening the door to more scientific breakthroughs

Evaluation of serial crystallographic structure determination within megahertz pulse trains

The new European X-ray Free-Electron Laser (European XFEL) is the first X-ray free-electron laser capable of delivering intense X-ray pulses with a megahertz interpulse spacing in a wavelength range suitable for atomic resolution structure determination. An outstanding but crucial question is whether the use of a pulse repetition rate nearly four orders of magnitude higher than previously possible results in unwanted structural changes due to either radiation damage or systematic effects on data quality. Here, separate structures from the first and subse- quent pulses in the European XFEL pulse train were determined, showing that there is essentially no difference between structures deter- mined from different pulses under currently available operating conditions at the European XFEL