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Additional file 1: Fig. S1. Fragments used as a basis for the virtual screening. Table S1. 99th percentile of TransFS and SuCOS scores per fragment. Fig. S2. Top scoring compounds by dcTMD. Table S2. Links for accessing the workflows.

Over 115 participants, including researchers, managers and IT professionals from the photon and neutron communities and representatives of the European Commission, attended the 2nd online Photon and Neutron EOSC Symposium, jointly organised by PaNOSC and ExPaNDS on 26 October 2021. Find in this booklet all presentations, the recordings will also be made available shortly.

This deliverable 3.2 is a report on the demonstrator projects, the eight scientific and technical pilot projects that were selected to provide concrete scientific use-cases and guide and structure the work done in EOSC-Life to build an open digital and collaborative space for biological and medical research. We report in this deliverable the process of integration of the demonstrators within EOSC-Life, the achievement of the demonstrators who developed and made available to the scientific community several valuable resources (databases, workflows, web platform...), the actions undertaken within EOSC-Life to disseminate the demonstrator achievement and finally the results of the demonstrator survey to learn from the demonstrator experience and improve the integration of the new pilot project within EOSC-Life.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

In an effort to characterise the human NUDIX family SGC Oxford has expressed recombinant human NUDT7 as part of the SGC chemical probe programme and solved the first crystal structure of this enzyme. This enabled a crystallographic fragment screen which in conjunction with a separate covalent fragment approach yielded a first-in-class small molecule inhibitor of NUDT7 with activity in the single-digit micromolar range in a catalytic assay. This compound paves the way for chemical probe development and further functional exploration of NUDT7 in physiological and disease contexts.

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

An alternative strategy for PEG sampling is suggested through the use of four newly defined PEG smears to enhance chemical space in reduced screens with a benefit towards protein crystallization., The quest for an optimal limited set of effective crystallization conditions remains a challenge in macromolecular crystallography, an issue that is complicated by the large number of chemicals which have been deemed to be suitable for promoting crystal growth. The lack of rational approaches towards the selection of successful chemical space and representative combinations has led to significant overlapping conditions, which are currently present in a multitude of commercially available crystallization screens. Here, an alternative approach to the sampling of widely used PEG precipitants is suggested through the use of PEG smears, which are mixtures of different PEGs with a requirement of either neutral or cooperatively positive effects of each component on crystal growth. Four newly defined smears were classified by molecular-weight groups and enabled the preservation of specific properties related to different polymer sizes. These smears not only allowed a wide coverage of properties of these polymers, but also reduced PEG variables, enabling greater sampling of other parameters such as buffers and additives. The efficiency of the smear-based screens was evaluated on more than 220 diverse recombinant human proteins, which overall revealed a good initial crystallization success rate of nearly 50%. In addition, in several cases successful crystallizations were only obtained using PEG smears, while various commercial screens failed to yield crystals. The defined smears therefore offer an alternative approach towards PEG sampling, which will benefit the design of crystallization screens sampling a wide chemical space of this key precipitant.

The productive exploration of chemical space is an enduring challenge in chemical biology and medicinal chemistry. Natural products are biologically relevant, and their frameworks have facilitated chemical tool and drug discovery. A “top‐down” synthetic approach is described that enabled a range of complex bridged intermediates to be converted with high step efficiency into 26 diverse sp3‐rich scaffolds. The scaffolds have local natural product‐like features, but are only distantly related to specific natural product frameworks. To assess biological relevance, a set of 52 fragments was prepared, and screened by high‐throughput crystallography against three targets from two protein families (ATAD2, BRD1 and JMJD2D). In each case, 3D fragment hits were identified that would serve as distinctive starting points for ligand discovery. This demonstrates that frameworks that are distantly related to natural products can facilitate discovery of new biologically relevant regions within chemical space.

Summary Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design., Highlights • Structures of human and bacterial MCDs were determined at up to 2.3 Å resolution • Distinct tetrameric and dimeric MCD oligomerizations were observed • Unexpected homology to the GNAT superfamily gives insights into catalytic mechanism • The structures provide the molecular basis for the disease-causing mutations in MCD, Malonyl-CoA decarboxylase (MCD) is important in fatty acid metabolism. Froese et al. report structures of several MCDs and show that the MCD catalytic domain shares structural homology with GNAT superfamily. The structures further our understanding of catalysis, pathogenic mutations, and drug design.

Escherichia coli ketopantoate reductase (KPR), encoded by the gene panE, is an NADPH dependent oxidoreductase which catalyses the reduction of ketopantoate to pantoate, a step in the biosynthetic pathway of pantothenate (vitamin B5). KPR is the least well-characterised enzyme of the pathway because of the presence of another enzyme which can carry out the same reaction,1 acetohydroxyisomeroreductase, an enzyme from valine biosynthesis. In order to understand the catalytic mechanism of KPR we overexpressed and purified it and solved the crystal structure. The structure of KPR was solved by a selenomethionine MAD experiment at beamline BW7A at the EMBL outstation in DESY, Hamburg. Data were collected to 2.4 A at 3 wavelengths, selected from an X-ray fluorescence spectrum to optimise the anomalous signal. Selenium sites were found with the program SHELXS96 and data phased with SHARP using all three wavelength data sets. Experimental phases were improved by solvent flattening using Solomon, with a solvent content of 43%. The final electron-density map was easily interpretable. Native data were collected to 1.7 A at Daresbury SRS beamline 9.6. KPR crystals are tetragonal, space group P42212 (a = 104.2 and c = 55.8 A), with one molecule in the asymmetric unit. The enzyme is monomeric and has two domains. The N-terminal domain has an alpha-beta fold of Rossman type, while the C-terminal domain is all alpha helical. There is no evidence of bound cofactor (NADPH). Comparison of the fold against all structures in the Protein Data Bank using the DALI server revealed that it is similar to norvaline dehydrogenase.2 _____________ [1] M. E. Frodyma and D. Downs, J. Biol. Chem 273 (1998) 5572. [2] K. L. Britton, Y. Asano and D. W. Rice, Nature Struct. Biol. 5 (1998) 593.

Compound availability is a critical property for design prioritization across the drug discovery pipeline. Historically, and despite their multiple limitations, compound-oriented synthetic accessibility scores have been used as proxies for this problem. However, the size of the catalogues of commercially available molecules has dramatically increased over the last decade, redefining the problem of compound accessibility as a matter of budget. In this paper we show that if compound prices are the desired proxy for compound availability, then synthetic accessibility scores are not effective strategies for us in selection. Our approach, CopriNet, is a retrosynthesis-free deep learning model trained on 2D graph representations of compounds alongside their prices extracted from the Mcule catalogue. We show that CoPriNet provides price predictions that correlate far better with actual compound prices than any synthetic accessibility score. Moreover, unlike standard retrosynthesis methods, CoPriNet is rapid, with execution times comparable to popular synthetic accessibility metrics, and thus is suitable for high-throughput experiments including virtual screening and de novo compound generation. While the Mcule catalogue is a proprietary dataset, the CoPriNet source code and the model trained on the proprietary data as well as the fraction of the catalogue (100K compound/prices) used as test dataset have been made publicly available at https://github.com/oxpig/CoPriNet.

The pleckstrin homology domain interacting protein (PHIP) is a multidomain protein that is involved in cell morphology and cytoskeletal organization. It is suggested that PHIP regulates insulin-like growth factor signalling pathways. Suppression of its expression inhibits melanoma, breast, and lung tumour cell proliferation and invasion making it a drug target of choice [1]. The protein is composed of 8 WD repeats, which are known to fold into a ß-propeller domain, and two bromodomains. The second bromodomain of PHIP (PHIP2) is the only domain that has been structurally characterized. Bromodomains participate in gene expression modulation through the binding of acetylated lysine containing peptides located on histone tails. Multiple probes and drug-candidates have been designed to inhibit these proteins [2]. A highly reproducible and well-diffracting PHIP2 crystal was obtained in a C2 space group, thereby providing necessary material for an XChem [3] high-throughput crystallographic fragment screening experiment. Crystals were are soaked with 799 fragments, fished and shot in a semi-supervised and high-throughput fashion resulting in hundreds of data sets. Of these, 47 hits were identified at the pharmacologically relevant acetylated lysine binding site. This was done using the PanDDa4 method that takes advantage of the abundance of datasets to perform statistical extraction of weak binding events. The resulting structures were used as a challenging data set for the 7th edition of the SAMPL challenge for protein-ligands. The challenge was divided into 3 stages: First, participants were tasked to predict binders from non-binders. In the second stage, participants were asked to predict the poses of all 47 known fragments. Lastly, participants were asked to mine a database and propose follow-up compounds that would bind. Only a small number of groups participated, and their predictions were rather inaccurate, highlighting the sense that predictions on fragment-protein complexes appear to be harder than for larger, more drug-like compounds. We discuss the implications of these results and consider where the community should focus its efforts in the future. [1] de Semir, D., Bezrookove, V., Nosrati, M., Dar, A., Wu, C., Shen, J., Rieken, C., Venkatasubramanian, M., Miller, J., Desprez, P., McAllister, S., Soroceanu, L., Debs, R., Salomonis, N., Schadendorf, D., Cleaver, J. and Kashani-Sabet, M., 2018. PHIP as a therapeutic target for driver-negative subtypes of melanoma, breast, and lung cancer. Proceedings of the National Academy of Sciences, 115(25), pp.E5766-E5775. [2] Cochran, A., Conery, A. and Sims, R., 2019. Bromodomains: a new target class for drug development. Nature Reviews Drug Discovery, 18(8), pp.609-628. [3] Douangamath, A., Fearon, D., Gehrtz, P., Krojer, T., Lukacik, P., Owen, C., Resnick, E., Strain-Damerell, C., Aimon, A., Ábrányi-Balogh, P., Brandão-Neto, J., Carbery, A., Davison, G., Dias, A., Downes, T., Dunnett, L., Fairhead, M., Firth, J., Jones, S., Keeley, A., Keserü, G., Klein, H., Martin, M., Noble, M., O’Brien, P., Powell, A., Reddi, R., Skyner, R., Snee, M., Waring, M., Wild, C., London, N., von Delft, F. and Walsh, M., 2020. Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease. Nature Communications, 11(1). [4] Pearce, N., Krojer, T., Bradley, A., Collins, P., Nowak, R., Talon, R., Marsden, B., Kelm, S., Shi, J., Deane, C. and von Delft, F., 2017. A multi-crystal method for extracting obscured crystallographic states from conventionally uninterpretable electron density. Nature Communications, 8(1).

Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10–14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery., Fragment-based screening of a shape-diverse collection yielded four hits against three proteins. Up to 14 analogues of each hit were rapidly generated, enabling four fragment growth vectors to be explored using inexpensive materials and reliable synthetic transformations.