Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket.

To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography.

Fast fragment and compound screening pipeline at the Swiss Light Source.

Crystallography-based fragment screening is a highly effective technique employed in structure-based drug discovery to expand the range of lead development opportunities. It allows screening and sorting of weakly binding, low molecular mass fragments, which can be developed into larger high-affinity lead compounds. Technical improvements at synchrotron beamlines, design of innovative libraries mapping chemical space efficiently, effective soaking methods and enhanced data analysis have enabled the implementation of high-throughput fragment screening pipelines at multiple synchrotron facilities. This widened access to CBFS beyond the pharma industry has allowed academic users to rapidly screen large quantities of fragment-soaked protein crystals. The positive outcome of a CBFS campaign is a set of structures that present the three-dimensional arrangement of fragment-protein complexes in detail, thereby providing information on the location and the mode of interaction of bound fragments. Through this review, we provide users with a comprehensive guide that sets clear expectations before embarking on a crystallography-based fragment screening campaign. We present a list of essential pre-requirements that must be assessed, including the suitability of your current crystal system for a fragment screening campaign. Furthermore, we extensively discuss the available methodological options, addressing their limitations and providing strategies to overcome them. Additionally, we provide a brief perspective on how to proceed once hits are obtained. Notably, we emphasize the solutions we have implemented for instrumentation and software development within our Fast Fragment and Compound Screening pipeline. We also highlight third-party software options that can be utilized for rapid refinement and hit assessment.

SDU - software for high-throughput automated data collection at the Swiss Light Source.

Recent advances in automation have fostered the development of unattended data collection services at a handful of synchrotron facilities worldwide. At the Swiss Light Source, the installation of new high-throughput sample changers at all three macromolecular crystallography beamlines and the commissioning of the Fast Fragment and Compound Screening pipeline created a unique opportunity to automate data acquisition. Here, the DA+ microservice software stack upgrades, implementation of an automatic loop-centering service and deployment of the Smart Digital User (SDU) software for unattended data collection are reported. The SDU software is the decision-making software responsible for communications between services, sample and device safety, sample centering, sample alignment with grid based X-ray diffraction and, finally, data collection.

Probing ligand binding of endothiapepsin by `temperature-resolved' macromolecular crystallography.

Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery.

Fast fragment- and compound-screening pipeline at the Swiss Light Source.

Over the last two decades, fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds. X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods. However, it is also often used with great success as a primary screening technique. In recent years, technological advances at macromolecular crystallography beamlines in terms of instrumentation, beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography. Here, the development of the Fast Fragment and Compound Screening (FFCS) platform, an integrated next-generation pipeline for crystal soaking, handling and data collection which allows crystallography-based screening of protein crystals against hundreds of fragments and compounds, at the Swiss Light Source is reported.

Combining High-Throughput Synthesis and High-Throughput Protein Crystallography for Accelerated Hit Identification.

Protein crystallography (PX) is widely used to drive advanced stages of drug optimization or to discover medicinal chemistry starting points by fragment soaking. However, recent progress in PX could allow for a more integrated role into early drug discovery. Here, we demonstrate for the first time the interplay of high throughput synthesis and high throughput PX. We describe a practical multicomponent reaction approach to acrylamides and -esters from diverse building blocks suitable for mmol scale synthesis on 96-well format and on a high-throughput nanoscale format in a highly automated fashion. High-throughput PX of our libraries efficiently yielded potent covalent inhibitors of the main protease of the COVID-19 causing agent, SARS-CoV-2. Our results demonstrate, that the marriage of in situ HT synthesis of (covalent) libraires and HT PX has the potential to accelerate hit finding and to provide meaningful strategies for medicinal chemistry projects.

Comprehensive Analysis of Binding Sites in Tubulin.

Tubulin plays essential roles in vital cellular activities and is the target of a wide range of proteins and ligands. Here, using a combined computational and crystallographic fragment screening approach, we addressed the question of how many binding sites exist in tubulin. We identified 27 distinct sites, of which 11 have not been described previously, and analyzed their relationship to known tubulin-protein and tubulin-ligand interactions. We further observed an intricate pocket communication network and identified 56 chemically diverse fragments that bound to 10 distinct tubulin sites. Our results offer a unique structural basis for the development of novel small molecules for use as tubulin modulators in basic research applications or as drugs. Furthermore, our method lays down a framework that may help to discover new pockets in other pharmaceutically important targets and characterize them in terms of chemical tractability and allosteric modulation.

Structural Insights into Plasticity and Discovery of Remdesivir Metabolite GS-441524 Binding in SARS-CoV-2 Macrodomain.

The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in the host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus targeting this protein domain may offer a therapeutic strategy to combat current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally occurring nucleotides, small molecules, and nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and provide chemical starting points for future inhibitor development.

Advances in long-wavelength native phasing at X-ray free-electron lasers.

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.

Selectively Disrupting m6A-Dependent Protein-RNA Interactions with Fragments.

We report a crystallographic analysis of small-molecule ligands of the human YTHDC1 domain that recognizes N6-methylated adenine (m6A) in RNA. The 30 binders are fragments (molecular weight < 300 g mol-1) that represent 10 different chemotypes identified by virtual screening. Despite the structural disorder of the binding site loop (residues 429-439), most of the 30 fragments emulate the two main interactions of the -NHCH3 group of m6A. These interactions are the hydrogen bond to the backbone carbonyl of Ser378 and the van der Waals contacts with the tryptophan cage. Different chemical groups are involved in the conserved binding motifs. Some of the fragments show favorable ligand efficiency for YTHDC1 and selectivity against other m6A reader domains. The structural information is useful for the design of modulators of m6A recognition by YTHDC1.