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.

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 nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

beamline for macromolecular crystallography (1). The photon energy be between 5.5 - 28 keV with the possibility of using a CdTe-detector for higher energies (> 22 keV). Beam are between 200 μm and 4 x 9 μm with a maximum photon flux of 1e13 ph/s at keV. P11 optimized high-throughput crystallography. 16M and cycle less than 2 min can be The automatic sample changer at P11 is based on the unipuck format with a total capacity of 23 pucks (368 samples) and a mounting cycle of s. the graphical user interface; full integration of tapedrive experiments is in progress. OnDA (6) is available for real time evaluation of SSX data and implementation of real-time SSX processing is in progress within an LTP.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the host’s innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 Å and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 Å, show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, 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.

Here we present the crystal structure of SARS-CoV-2 main protease (M pro ) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of M pro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of M pro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by M pro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by M pro , followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.

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 two repurposing drug libraries against the SARS-CoV-2 main protease (M pro ), which is essential for the virus replication and, thus, a potent drug target. 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 M pro . In subsequent cell-based viral reduction assays, one peptidomimetic and five 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.

Investigating metal organic systems with time-resolved photocrystallography poses a unique challenge while interpreting the time dependent photodifference maps. In these difference Fourier maps, the signals correspond to the movement of heavy metal atoms always overpower the signals from much lighter atoms attached to them. For a systematic assessment of the quality of the photodifference maps obtained from metal organic systems, in this work, LaueUtil and PRECOGNITION software were used to treat time-resolved Laue crystallography data of a [2x2] matrix-like Fe(II) complex. The rigid spot identification method in LaueUtil allows to identify and index > 250,000 reflections per 10 datasets. Though this leads to low completeness (< 30 software only treats the information from highly reliable diffraction spots. As a result, clean photodifference maps and small values in the thermal scale factor have been obtained. In the PRECOGNITION case, the package indexed more than 160,000 reflections per dataset. The resulting completeness is higher (>86 treatment. However, the dependence on the refinement of the lambda curve as well as the degradation of the sample may be reflected on the large thermal scale factors which also contribute as noise in the photodifference maps.

This paper reports a new packing type of α-cyclodextrin inclusion complexes, obtained here with succinic acid under low-temperature crystallization conditions. The structure of the 1:1 complex is characterized by heavy disorder of the guest, the solvent, and part of the host. The crystal packing belongs to the known channel-type structure; the basic structural unit is composed of cyclodextrin trimers, as opposed to the known isolated molecular or dimeric constructs, packed along the c-axis. Each trimer is made of crystallographically independent molecules assembled in a stacked vase-like cluster. A multi-temperature single-crystal X-ray diffraction analysis reveals the presence of dynamic disorder.

The structural behavior a long-chain imidazolium-based ionic liquid [C(10)mim]Cl-water mixtures has been HP investigated in detail by low-teruperature arid high-pressure crystallization methods, and the solid forms have been fully characterized by single-crystal X-ray diffraction. Form I, a monohydrate, crystallizes from solutions containing 6-8% (w/w) water; its structure exhibits bilayers, in which the cations alternate from layer to layer to create hydrophobic and hydrophilic regions. Form II, which is a Z' > 1 structure, incorporates a variable amount of water content (from pseudo-hemihydrate to pseudo-monohydrate) depending on the sample treatment and environment; this form crystallizes from solutions containing < ca. 6% (w/w) water and exhibits a double bilayered structure characterized by ordered and disordered regions. Forms I and II have been obtained at both low-temperature and high-pressure conditions. Form III is a trihydrate that has been exclusively obtained under high-pressure conditions above 0.55 GPa from solutions containing >80% (w/w) water. This structure is closely related -to that of-form I. While homeotypic and isotypic structures of forms I and II have been previously reported, form III represents, to the best of our knowledge, the only example of a highly hydrated long-chain imidazolium-based ionic liquid isolated in the solid state. The formation of mesophases in the title compound has also been investigated by polarized optical microscopy, and the results were correlated with previous liquid-crystal Studies on related compounds.

The solid-state polymorphism of the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim][PF6], has been investigated via low-temperature and high-pressure crystallisation experiments. The samples have been characterised by single-crystal X-ray diffraction, optical microscopy and Raman spectroscopy. The solid-state phase behaviour of the compound is confirmed and clarified with respect to previous phase diagrams. The structures of the previously reported γ-form, which essentially exhibits a G′T cation conformation, as well as those of the elusive β- and α-forms, are reported. Crystals of the β-phase are twinned and the structure is heavily disordered; the cation conformation in this form is predominantly TT, though significant contributions from other less frequently encountered conformers are also observed at low temperature and high pressure. The cation conformation in the α-form is GT; the presence of the G′T conformer at 193 K in this phase can be eliminated on cooling to 100 K. Whilst X-ray structural data are overall in good agreement with previous interpretations based on Raman and NMR studies, they also reveal a more subtle interplay of intermolecular interactions, which give rise to a wider range of conformers than previously considered.

The X-ray single-crystal structure of (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, commonly known as (+)-6-aminopenicillanic acid (C8H12N2O3S) and a precursor of a variety of semi-synthetic penicillins, has been determined from synchrotron data at 150 K. The structure represents an ordered zwitterion and the crystals are nonmerohedrally twinned. The crystal structure is composed of a three-dimensional network built by three charge-assisted hydrogen bonds between the ammonium and carboxylate groups. The complementary analysis of the crystal packing by the PIXEL method brings to light the nature and ranking of the energetically most stabilizing intermolecular interaction energies. In accordance with the zwitterionic nature of the structure, PIXEL lattice energy calculations confirm the predominance of the Coulombic term (-379.1 kJ mol(-1)) ahead of the polarization (-141.4 kJ mol(-1)), dispersion (-133.7 kJ mol(-1)) and repulsion (266.3 kJ mol(-1)) contributions.

Due to their tunable physical and chemical properties, Room Temperature Ionic liquids (RTILs) have found numerous applications as solvents in industry and the life sciences.The number of reported crystal structures of RTILs has been steadingly increasing; however, the phenomenon of polymorphism has been less widely investigated.Up to three crystalline phases of 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF 6 ]) under non-ambient conditions were previously characterized by DSC [1,2], Raman [1,3,4], and solid-state NMR spectroscopy [5] as well as powder X-ray diffraction [2]; in contrast, only one crystal structure was reported [6,7].We here present the single-crystal structures of all three polymorphs of this compound obtained under low-temperature and high-pressure crystallisation conditions.These polymorphs and their interconversion were also investigated by Raman spectroscopy and optical microscopy.By elucidating the conformations adopted by the cation and anion in the three structures, we clarify the polymorphic behaviour and previous phase diagrams of this compound.

The technique of high-pressure crystallisation from solution has been applied to the study of α-cyclodextrin hydrate formation. A previously unobserved hydrate, here termed Ib, has been obtained at 0.65 GPa and characterised by single-crystal X-ray diffraction using synchrotron radiation. This form exhibits a combination of structural features of other known hydrates and hydration states obtained by theoretical calculations but never found experimentally before.

As small molecules get larger [1], determining their structures becomes increasingly challenging.Initially, the problem is growing suitable crystals; while this can be a problem for even very small molecules, for bigger molecules it can be particularly challenging.Once that hurdle has been overcome data collection is the next difficulty, where solvent loss, and poor diffraction can be serious impediments.Synchrotron radiation can make the impossible possible, but can also introduce new problems for example, radiation damage.Finally, structure refinement is always difficult due problems including a paucity of data, the large number of parameters and often, large regions of poorly defined solvent.In the last 10 years, the author's view of what constitutes a large structure has changed significantly.Starting in a world where a unit cell contains only a handful of atoms, small pharmaceutical compunds could seem quite large.More recently, collaborators have found increasingly large molecules to study, the most recent being 11.5 Kda!Some of the stops on this Journey into the Unknown will be presented along with some of the pit-falls, obstacles, detours and cul-de-sacs: the Final Destination is unknown.

This thesis, which is divided into two parts, describes the use of non-ambient crystallisation techniques, in particular high-pressure, to explore on one hand inclusion complex formation of cyclodextrins with drug molecules (part 1), and on the other hand the solid-state behaviour of imidazolium-based ionic liquids (part 2). Much of this thesis is devoted to establishing detailed crystallisation protocols for the compounds studied and to reporting full structural analysis of the resulting crystals, which have been investigated by single-crystal X-ray diffraction using both laboratory and synchrotron sources. This work shows that the application of pressure has different effects on cyclodextrin-based inclusion complexes. When water is used as pressure transmitting medium, cyclodextrins undergo dissolution as function of increasing pressure, regardless of the inclusion state. Upon further pressurisation three events have been observed to take place: no crystallisation, crystallisation of host and guest molecules as separate entities or crystallisation of inclusion complexes. It has not been possible to rationalise the observed behaviour or predict which of the three events is most likely to occur for a given system. During the course of this work, novel inclusion complexes for α- and β- cyclodextrin have been obtained at both ambient- and high-pressure conditions; full structural characterisation has enabled to identify two novel packing motifs. As demonstrated for imidazolium-based ionic liquids, high-pressure is a powerful external factor for triggering crystal formation and phase transitions in this class of compounds. The solid-state behaviour of ionic liquids at high pressure has been correlated with the one at low temperature, proving that a thorough understanding and exploration of the crystallisation diagrams necessitates the use of both non-ambient techniques. The studies performed in this thesis demonstrate the importance of characterising crystalline phases by single-crystal X-ray diffraction methods. Thanks to the availability of accurate structural data it has been possible to unravel the elusive polymorphism of the most widely studied imidazolium-based ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate, which had been the subject of much debate and speculation in the literature. For 1-decyl-3-methylimidazolium chloride, structural data have enabled to distinguish between three distinct packing types of this ionic liquid in its hydrated forms, and to pin point subtle but important structural differences between the different crystalline phases.