Structural Elucidation and Antiviral Activity of Covalent Cathepsin L Inhibitors.

Emerging RNA viruses, including SARS-CoV-2, continue to be a major threat. Cell entry of SARS-CoV-2 particles via the endosomal pathway involves cysteine cathepsins. Due to ubiquitous expression, cathepsin L (CatL) is considered a promising drug target in the context of different viral and lysosome-related diseases. We characterized the anti-SARS-CoV-2 activity of a set of carbonyl- and succinyl epoxide-based inhibitors, which were previously identified as inhibitors of cathepsins or related cysteine proteases. Calpain inhibitor XII, MG-101, and CatL inhibitor IV possess antiviral activity in the very low nanomolar EC50 range in Vero E6 cells and inhibit CatL in the picomolar Ki range. We show a relevant off-target effect of CatL inhibition by the coronavirus main protease α-ketoamide inhibitor 13b. Crystal structures of CatL in complex with 14 compounds at resolutions better than 2 Å present a solid basis for structure-guided understanding and optimization of CatL inhibitors toward protease drug development.

SiteMine: Large-scale binding site similarity searching in protein structure databases.

Drug discovery and design challenges, such as drug repurposing, analyzing protein-ligand and protein-protein complexes, ligand promiscuity studies, or function prediction, can be addressed by protein binding site similarity analysis. Although numerous tools exist, they all have individual strengths and drawbacks with regard to run time, provision of structure superpositions, and applicability to diverse application domains. Here, we introduce SiteMine, an all-in-one database-driven, alignment-providing binding site similarity search tool to tackle the most pressing challenges of binding site comparison. The performance of SiteMine is evaluated on the ProSPECCTs benchmark, showing a promising performance on most of the data sets. The method performs convincingly regarding all quality criteria for reliable binding site comparison, offering a novel state-of-the-art approach for structure-based molecular design based on binding site comparisons. In a SiteMine showcase, we discuss the high structural similarity between cathepsin L and calpain 1 binding sites and give an outlook on the impact of this finding on structure-based drug design. SiteMine is available at https://uhh.de/naomi.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Online dynamic flat-field correction for MHz microscopy data at European XFEL.

The high pulse intensity and repetition rate of the European X-ray Free-Electron Laser (EuXFEL) provide superior temporal resolution compared with other X-ray sources. In combination with MHz X-ray microscopy techniques, it offers a unique opportunity to achieve superior contrast and spatial resolution in applications demanding high temporal resolution. In both live visualization and offline data analysis for microscopy experiments, baseline normalization is essential for further processing steps such as phase retrieval and modal decomposition. In addition, access to normalized projections during data acquisition can play an important role in decision-making and improve the quality of the data. However, the stochastic nature of X-ray free-electron laser sources hinders the use of standard flat-field normalization methods during MHz X-ray microscopy experiments. Here, an online (i.e. near real-time) dynamic flat-field correction method based on principal component analysis of dynamically evolving flat-field images is presented. The method is used for the normalization of individual X-ray projections and has been implemented as a near real-time analysis tool at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL.

Hard X-ray stereographic microscopy for single-shot differential phase imaging.

The characterisation of fast phenomena at the microscopic scale is required for the understanding of catastrophic responses of materials to loads and shocks, the processing of materials by optical or mechanical means, the processes involved in many key technologies such as additive manufacturing and microfluidics, and the mixing of fuels in combustion. Such processes are usually stochastic in nature and occur within the opaque interior volumes of materials or samples, with complex dynamics that evolve in all three dimensions at speeds exceeding many meters per second. There is therefore a need for the ability to record three-dimensional X-ray movies of irreversible processes with resolutions of micrometers and frame rates of microseconds. Here we demonstrate a method to achieve this by recording a stereo phase-contrast image pair in a single exposure. The two images are combined computationally to reconstruct a 3D model of the object. The method is extendable to more than two simultaneous views. When combined with megahertz pulse trains of X-ray free-electron lasers (XFELs) it will be possible to create movies able to resolve 3D trajectories with velocities of kilometers per second.

Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

X-ray structure of the Rhodobacter sphaeroides reaction center with an M197 Phe→His substitution clarifies the properties of the mutant complex.

The first steps of the global process of photosynthesis take place in specialized membrane pigment-protein complexes called photosynthetic reaction centers (RCs). The RC of the photosynthetic purple bacterium Rhodobacter sphaeroides, a relatively simple analog of the more complexly organized photosystem II in plants, algae and cyanobacteria, serves as a convenient model for studying pigment-protein interactions that affect photochemical processes. In bacterial RCs the bacteriochlorophyll (BChl) dimer P serves as the primary electron donor, and its redox potential is a critical factor in the efficient functioning of the RC. It has previously been shown that the replacement of Phe M197 by His strongly affects the oxidation potential of P (E m P/P+), increasing its value by 125 mV, as well as increasing the thermal stability of RC and its stability in response to external pressure. The crystal structures of F(M197)H RC at high resolution obtained using various techniques presented in this report clarify the optical and electrochemical properties of the primary electron donor and the increased resistance of the mutant complex to denaturation. The electron-density maps are consistent with the donation of a hydrogen bond from the imidazole group of His M197 to the C2-acetyl carbonyl group of BChl PB. The formation of this hydrogen bond leads to a considerable out-of-plane rotation of the acetyl carbonyl group and results in a 1.2 Šshift of the O atom of this group relative to the wild-type structure. Besides, the distance between BChl PA and PB in the area of pyrrole ring I was found to be increased by up to 0.17 Å. These structural changes are discussed in association with the spectral properties of BChl dimer P. The electron-density maps strongly suggest that the imidazole group of His M197 accepts another hydrogen bond from the nearest water molecule, which in turn appears to form two more hydrogen bonds to Asn M195 and Asp L155. As a result of the F(M197)H mutation, BChl PB finds itself connected to the extensive hydrogen-bonding network that pre-existed in wild-type RC. Dissimilarities in the two hydrogen-bonding networks near the M197 and L168 sites may account for the different changes of the E m P/P+ in F(M197)H and H(L168)F RCs. The involvement of His M197 in the hydrogen-bonding network also appears to be related to stabilization of the F(M197)H RC structure. Analysis of the experimental data presented here and of the data available in the literature points to the fact that the hydrogen-bonding networks in the vicinity of BChl dimer P may play an important role in fine-tuning the redox properties of the primary electron donor.

Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease.

The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral propagation and, additionally, dysregulation of the host innate immune system. Using a library of 40 potential metal-chelating compounds we performed an X-ray crystallographic screening against PLpro. As outcome we identified six compounds binding to the target protein. Here we describe the interaction of one hydrazone (H1) and five thiosemicarbazone (T1-T5) compounds with the two distinct natural substrate binding sites of PLpro for ubiquitin and ISG15. H1 binds to a polar groove at the S1 binding site by forming several hydrogen bonds with PLpro. T1-T5 bind into a deep pocket close to the polyubiquitin and ISG15 binding site S2. Their interactions are mainly mediated by multiple hydrogen bonds and further hydrophobic interactions. In particular compound H1 interferes with natural substrate binding by sterical hindrance and induces conformational changes in protein residues involved in substrate binding, while compounds T1-T5 could have a more indirect effect. Fluorescence based enzyme activity assay and complementary thermal stability analysis reveal only weak inhibition properties in the high micromolar range thereby indicating the need for compound optimization. Nevertheless, the unique binding properties involving strong hydrogen bonding and the various options for structural optimization make the compounds ideal lead structures. In combination with the inexpensive and undemanding synthesis, the reported hydrazone and thiosemicarbazones represent an attractive scaffold for further structure-based development of novel PLpro inhibitors by interrupting protein-protein interactions at the S1 and S2 site.

Analyzing structural features of proteins from deep-sea organisms.

Protein adaptations to extreme environmental conditions are drivers in biotechnological process optimization and essential to unravel the molecular limits of life. Most proteins with such desirable adaptations are found in extremophilic organisms inhabiting extreme environments. The deep sea is such an environment and a promising resource that poses multiple extremes on its inhabitants. Conditions like high hydrostatic pressure and high or low temperature are prevalent and many deep-sea organisms tolerate multiple of these extremes. While molecular adaptations to high temperature are comparatively good described, adaptations to other extremes like high pressure are not well-understood yet. To fully unravel the molecular mechanisms of individual adaptations it is probably necessary to disentangle multifactorial adaptations. In this study, we evaluate differences of protein structures from deep-sea organisms and their respective related proteins from nondeep-sea organisms. We created a data collection of 1281 experimental protein structures from 25 deep-sea organisms and paired them with orthologous proteins. We exhaustively evaluate differences between the protein pairs with machine learning and Shapley values to determine characteristic differences in sequence and structure. The results show a reasonable discrimination of deep-sea and nondeep-sea proteins from which we distinguish correlations previously attributed to thermal stability from other signals potentially describing adaptions to high pressure. While some distinct correlations can be observed the overall picture appears intricate.

Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals.

The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Šresolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.

Ptychographic X-ray speckle tracking with multi-layer Laue lens systems.

The ever-increasing brightness of synchrotron radiation sources demands improved X-ray optics to utilize their capability for imaging and probing biological cells, nano-devices and functional matter on the nanometre scale with chemical sensitivity. Hard X-rays are ideal for high-resolution imaging and spectroscopic applications owing to their short wavelength, high penetrating power and chemical sensitivity. The penetrating power that makes X-rays useful for imaging also makes focusing them technologically challenging. Recent developments in layer deposition techniques have enabled the fabrication of a series of highly focusing X-ray lenses, known as wedged multi-layer Laue lenses. Improvements to the lens design and fabrication technique demand an accurate, robust, in situ and at-wavelength characterization method. To this end, a modified form of the speckle tracking wavefront metrology method has been developed. The ptychographic X-ray speckle tracking method is capable of operating with highly divergent wavefields. A useful by-product of this method is that it also provides high-resolution and aberration-free projection images of extended specimens. Three separate experiments using this method are reported, where the ray path angles have been resolved to within 4 nrad with an imaging resolution of 45 nm (full period). This method does not require a high degree of coherence, making it suitable for laboratory-based X-ray sources. Likewise, it is robust to errors in the registered sample positions, making it suitable for X-ray free-electron laser facilities, where beam-pointing fluctuations can be problematic for wavefront metrology.

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

A crystallographic indexing algorithm, pinkIndexer, is presented for the analysis of snapshot diffraction patterns. It can be used in a variety of contexts including measurements made with a monochromatic radiation source, a polychromatic source or with radiation of very short wavelength. As such, the algorithm is particularly suited to automated data processing for two emerging measurement techniques for macromolecular structure determination: serial pink-beam X-ray crystallography and serial electron crystallography, which until now lacked reliable programs for analyzing many individual diffraction patterns from crystals of uncorrelated orientation. The algorithm requires approximate knowledge of the unit-cell parameters of the crystal, but not the wavelengths associated with each Bragg spot. The use of pinkIndexer is demonstrated by obtaining 1005 lattices from a published pink-beam serial crystallography data set that had previously yielded 140 indexed lattices. Additionally, in tests on experimental serial crystallography diffraction data recorded with quasi-monochromatic X-rays and with electrons the algorithm indexed more patterns than other programs tested.

Multimodal X-ray imaging of nanocontainer-treated macrophages and calcium distribution in the perilacunar bone matrix.

Studies of biological systems typically require the application of several complementary methods able to yield statistically-relevant results at a unique level of sensitivity. Combined X-ray fluorescence and ptychography offer excellent elemental and structural imaging contrasts at the nanoscale. They enable a robust correlation of elemental distributions with respect to the cellular morphology. Here we extend the applicability of the two modalities to higher X-ray excitation energies, permitting iron mapping. Using a long-range scanning setup, we applied the method to two vital biomedical cases. We quantified the iron distributions in a population of macrophages treated with Mycobacterium-tuberculosis-targeting iron-oxide nanocontainers. Our work allowed to visualize the internalization of the nanocontainer agglomerates in the cytosol. From the iron areal mass maps, we obtained a distribution of antibiotic load per agglomerate and an average areal concentration of nanocontainers in the agglomerates. In the second application we mapped the calcium content in a human bone matrix in close proximity to osteocyte lacunae (perilacunar matrix). A concurrently acquired ptychographic image was used to remove the mass-thickness effect from the raw calcium map. The resulting ptychography-enhanced calcium distribution allowed then to observe a locally lower degree of mineralization of the perilacunar matrix.

Co-crystal structure of the iMango-III fluorescent RNA aptamer using an X-ray free-electron laser.

Turn-on aptamers are in vitro-selected RNAs that bind to conditionally fluorescent small molecules and enhance their fluorescence. Upon binding TO1-biotin, the iMango-III aptamer achieves the largest fluorescence enhancement reported for turn-on aptamers (over 5000-fold). This aptamer was generated by structure-guided engineering and functional reselection of the parental aptamer Mango-III. Structures of both Mango-III and iMango-III have previously been determined by conventional cryocrystallography using synchrotron X-radiation. Using an X-ray free-electron laser (XFEL), the room-temperature iMango-III-TO1-biotin co-crystal structure has now been determined at 3.0 Šresolution. This structural model, which was refined against a data set of ∼1300 diffraction images (each from a single crystal), is largely consistent with the structures determined from single-crystal data sets collected at 100 K. This constitutes a technical benchmark on the way to XFEL pump-probe experiments on fluorescent RNA-small molecule complexes.

On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies.

Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, 'naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

Co-crystal structure of the Fusobacterium ulcerans ZTP riboswitch using an X-ray free-electron laser.

Riboswitches are conformationally dynamic RNAs that regulate gene expression by binding specific small molecules. ZTP riboswitches bind the purine-biosynthetic intermediate 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP) and its triphosphorylated form (ZTP). Ligand binding to this riboswitch ultimately upregulates genes involved in folate and purine metabolism. Using an X-ray free-electron laser (XFEL), the room-temperature structure of the Fusobacterium ulcerans ZTP riboswitch bound to ZMP has now been determined at 4.1 Šresolution. This model, which was refined against a data set from ∼750 diffraction images (each from a single crystal), was found to be consistent with that previously obtained from data collected at 100 K using conventional synchrotron X-radiation. These experiments demonstrate the feasibility of time-resolved XFEL experiments to understand how the ZTP riboswitch accommodates cognate ligand binding.

Localising functionalised gold-nanoparticles in murine spinal cords by X-ray fluorescence imaging and background-reduction through spatial filtering for human-sized objects.

Accurate in vivo localisation of minimal amounts of functionalised gold-nanoparticles, enabling e.g. early-tumour diagnostics and pharmacokinetic tracking studies, requires a precision imaging system offering very high sensitivity, temporal and spatial resolution, large depth penetration, and arbitrarily long serial measurements. X-ray fluorescence imaging could offer such capabilities; however, its utilisation for human-sized scales is hampered by a high intrinsic background level. Here we measure and model this anisotropic background and present a spatial filtering scheme for background reduction enabling the localisation of nanoparticle-amounts as reported from small-animal tumour models. As a basic application study towards precision pharmacokinetics, we demonstrate specific localisation to sites of disease by adapting gold-nanoparticles with small targeting ligands in murine spinal cord injury models, at record sensitivity levels using sub-mm resolution. Both studies contribute to the future use of molecularly-targeted gold-nanoparticles as next-generation clinical diagnostic and pharmacokinetic tools.

Demonstration of femtosecond X-ray pump X-ray probe diffraction on protein crystals.

The development of X-ray free-electron lasers (XFELs) has opened the possibility to investigate the ultrafast dynamics of biomacromolecules using X-ray diffraction. Whereas an increasing number of structures solved by means of serial femtosecond crystallography at XFELs is available, the effect of radiation damage on protein crystals during ultrafast exposures has remained an open question. We used a split-and-delay line based on diffractive X-ray optics at the Linac Coherent Light Source XFEL to investigate the time dependence of X-ray radiation damage to lysozyme crystals. For these tests, crystals were delivered to the X-ray beam using a fixed-target approach. The presented experiments provide probe signals at eight different delay times between 19 and 213 femtoseconds after a single pump event, thereby covering the time-scales relevant for femtosecond serial crystallography. Even though significant impact on the crystals was observed at long time scales after exposure with a single X-ray pulse, the collected diffraction data did not show significant signal reduction that could be assigned to beam damage on the crystals in the sampled time window and resolution range. This observation is in agreement with estimations of the applied radiation dose, which in our experiment was clearly below the values expected to cause damage on the femtosecond time scale. The experiments presented here demonstrate the feasibility of time-resolved pump-multiprobe X-ray diffraction experiments on protein crystals.

Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene.

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a free-standing graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 Å and 2.4 Å resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects.

X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography.

Specific radiation damage can be used to determine phases de novo from macromolecular crystals. This method is known as radiation-damage-induced phasing (RIP). One limitation of the method is that the dose of individual data sets must be minimized, which in turn leads to data sets with low multiplicity. A solution to this problem is to use data from multiple crystals. However, the resulting signal can be degraded by a lack of isomorphism between crystals. Here, it is shown that serial synchrotron crystallography in combination with selective merging of data sets can be used to determine high-quality phases for insulin and thaumatin, and that the increased multiplicity can greatly enhance the success rate of the experiment.