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CRISPR/Cas9 has revolutionized several areas of life science; however, methods to control the Cas9 activity are needed for both scientific and therapeutic applications. Anti-CRISPR proteins are known to inhibit the CRISPR/Cas adaptive immunity; however, in vivo delivery of such proteins is problematic. Instead, small-molecule Cas9 inhibitors could serve as useful tools due to their permeable, proteolytically stable, and non-immunogenic nature. Here, we identified a small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughput screening utilizing an Escherichia coli selection system. Extensive structure-activity relationship studies, which involved a deconstruction-reconstruction strategy, resulted in a range of analogues with significant improvements in the inhibitory activity. Based on NMR and electrophoretic mobility shift assays, we propose that the inhibitory action of these compounds likely results from direct binding to apo-Cas9, preventing Cas9:gRNA complex formation. These molecules may find use as Cas9 modulators in various applications.

The synthesis of a fully deprotected Kdo-containing rhamnogalacturonan II pentasaccharide is described. The strategy relies on the preparation of a suitably protected homogalacturonan tetrasaccharide backbone through a post-glycosylation oxidation approach, and its stereoselective glycosylation with a Kdo fluoride donor.

Often referred to as the “Swiss Army knife” of the immune system, invariant natural killer T (iNKT) cells are a subpopulation of T lymphocytes stimulated by the synthetic glycolipid α-galactosylceramide (αGalCer) when in complex with the CD1d receptor of antigen presenting cells. Through their ability to produce TH1 and TH2 cytokines and co-stimulate several other lymphocytes, iNKT cells have emerged as central players in directing the immune response in a range of physiological processes, such as infections, autoimmune diseases, and cancer. Over the years, synthetic chemistry has advanced the field of iNKT cell stimulation with the development of more efficient approaches to prepare αGalCer, and, additionally, with the chemical synthesis of αGalCer analogs in the search of better TH1/TH2 cytokine skewing compounds for therapeutic applications. Here we review the strategies for the synthesis of αGalCer and its analogs, including synthetic probes, together with the most important advances in the understanding of the mechanism of action of these compounds, as a guide to the available tools for interrogating the iNKT cell–αGalCer–CD1d complex and inspiration for future research.

Azoxy compounds belong to a small group of natural products sharing a common functional group with the general structure RN = N+(O-)R. Three new azoxides, azodyrecins A-C (1-3), were isolated from a soil-derived Streptomyces sp. strain P8-A2. The cis-alkenyl unit in 1-3 was found to readily isomerize to the trans-congeners (4-6). The structures of the new compounds were determined by detailed spectroscopic (1D/2D NMR) and HRMS data analysis. Azodyrecins belong to a new class of natural azoxy compounds and are proposed to derive from l-alanine and alkylamines. The absolute configurations of 1-6 were defined by comparison of ECD spectra. While no antimicrobial effects were observed for 1 against Staphylococcus aureus, Vibrio anguillarum, or Candida albicans, azodyrecin B (2) exhibited cytotoxicity against the human leukemia cell line HL-60 with an IC50 value of 2.2 μM.

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array‐based assay for the high‐throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl‐, fucosyl‐, and xylosyltransferases can transfer azido‐functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3‐dipolar cycloaddition reaction with an alkynyl‐modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research., Express and screen: A high‐throughput assay based on the application of azido‐functionalized sugar nucleotide donors on a synthetic glycan array for screening heterologously expressed plant glycosyltransferases was developed. The opportunity to express and screen large numbers of glycosyltransferases instead of rationally selected candidates will markedly accelerate the elucidation of plant cell wall biosynthetic pathways.

S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid structural and functional studies of carbohydrate-active enzymes, as they are often inert or act as enzyme inhibitors. In this context, this work focuses on the introduction of an S-linkage into arabinoxylan oligosaccharides (AXs) in order to obtain a small collection of synthetic tools for the study of AXs degrading enzymes. The key step for the introduction of the S-glycosidic linkage involved anomeric thiol S-alkylation of an orthogonally protected L-arabinopyranoside triflate. The resulting S-linked disaccharide was subsequently employed in a series of glycosylation reactions to obtain a selectively protected tetrasaccharide. This could be further elaborated through chemoselective deprotection and glycosylation reactions to introduce branching L-arabinofuranosides.

The fungal endophyte

Arabinogalactan proteins are proteoglycans located in the plant cell wall. Most arabinogalactan proteins are composed of carbohydrate moieties of β-(1→3)-galactan main chains with β-(1→6)-galactan side chains terminated by other glycans. In this study, three novel endo-β-(1→3)-galactanases were identified and the substrate specificity was further studied using well-defined galactan oligomers. Linear and branched β-(1→3)-linked galactans, which resemble the carbohydrate core of the arabinogalactan protein, were used for the characterization of endo-β-(1→3)-galactanases. The identified enzymes required at least three consecutive galactose residues for activity. Non-substituted regions were preferred, but substituents in the -2 and +2 and in some cases also -1 and +1 subsites were tolerated to some extent, depending on the branching pattern, however at a significantly lower rate/frequency.

Rapid development within the fields of both fragment-based drug discovery (FBDD) and medicinal targeting of RNA provides possibilities for combining technologies and methods in novel ways. This review provides an overview of fragment-based screening (FBS) against RNA targets, including a discussion of the most recently used screening and hit validation methods such as NMR spectroscopy, X-ray crystallography, and virtual screening methods. A discussion of fragment library design based on research from small-molecule RNA binders provides an overview on both the currently limited guidelines within RNA-targeting fragment library design, and future possibilities. Finally, future perspectives are provided on screening and hit validation methods not yet used in combination with both fragment screening and RNA targets.

The plant cuticle is deposited on the surface of primary plant organs, such as leaves, fruits, and floral organs, forming a diffusion barrier and protecting the plant against various abiotic and biotic stresses. Cutin, the structural polyester of the plant cuticle, is synthesized in the apoplast. Plasma-membrane-localized ATP-binding cassette (ABC) transporters of the G family have been hypothesized to export cutin precursors. Here, we characterize SlABCG42 of tomato representing an ortholog of AtABCG32 in Arabidopsis. SlABCG42 expression in Arabidopsis complements the cuticular deficiencies of the Arabidopsis pec1/abcg32 mutant. RNAi-dependent downregulation of both tomato genes encoding proteins highly homologous to AtABCG32 (SlABCG36 and SlABCG42) leads to reduced cutin deposition and formation of a thinner cuticle in tomato fruits. By using a tobacco (Nicotiana benthamiana) protoplast system, we show that AtABCG32 and SlABCG42 have an export activity for 10,16-dihydroxy hexadecanoyl-2-glycerol, a cutin precursor in vivo. Interestingly, also free ω-hydroxy hexadecanoic acid as well as hexadecanedioic acid were exported, furthering the research on the identification of cutin precursors in vivo and the respective mechanisms of their integration into the cutin polymer.

Reactive oxygen species (ROS) are involved in physiological cellular processes including differentiation, proliferation, and apoptosis by acting as signaling molecules or regulators of transcription factors. The maintenance of appropriate cellular ROS levels is termed redox homeostasis, a balance between their production and neutralization. High concentrations of ROS may contribute to severe pathological events including cancer, neurodegenerative, and cardiovascular diseases. In recent years, approaches to target the sources of ROS production directly in order to develop tool compounds or potential therapeutics have been explored. Herein, we briefly outline the major sources of cellular ROS production and comprehensively review the targeting of these by small-molecule inhibitors. We critically assess the value of ROS inhibitors with different mechanisms-of-action, including their potency, mode-of-action, known off-target effects, and clinical or preclinical status, while suggesting future avenues of research in the field.

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The Arabidopsis thaliana genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI Arabidopsis GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-com-patible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (Nicotiana benthamiana) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identify-ing effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic accep-tors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal4) and β-1,3-galacto-pentose (β-1,3-Gal5) to microsomes expressing individual GT14 enzymes, we verified the β-glucu-ronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In ad-dition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosyn-thesis for these GT14 members. Together, the updated Arabidopsis GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.

The fungal endophyte Aspergillus sp. strain FVL2, isolated from the traditional medicinal fennel plant, Foeniculum vulgare, was investigated for secondary metabolites. Fermentation on rice medium followed by chromatographic separation delivered three new natural products, 7-demethyl-neosulochrin (1), fumigaclavine I (3) and N-benzoyl-tryptophan (6) together with further 14 known metabolites, 1-O-methyl-sulochrin-4'-sulfate, questin, laccaic acid, isorhodoptilometrin, fumigaclavine A, fumigaclavine C, fumitremorgin C, fumigaquinazoline C, tryptoquivaline J, trypacidin, 3'-O-demethyl-sulochrin, 1-O-methyl-sulochrin, protocatechuic acid, and vermelone. The chemical structures of the new metabolites were determined by NMR spectroscopy and ESI HR mass spectrometry. For fumigaclavine I, we observed the partial deuterium transfer from the solvent to the enol form with a remarkable high stereo selectivity. The discovery of the new seco-anthraquinone 7-demethyl-neosulochrin (1) revealed a second type of ring cleavage by a questin oxygenase. The discovery of diverse secondary metabolites broadens the chemical space of Aspergillus.

Background Endo-β-1,4-galactanases are glycoside hydrolases (GH) from the GH53 family belonging to the largest clan of GHs, clan GH-A. GHs are ubiquitous and involved in a myriad of biological functions as well as being widely used industrially. Endo-β-1,4-galactanases, in particular hydrolyse galactan and arabinogalactan in pectin, a major component of the primary plant cell wall, with important functions in plant defence and application in the food and other industries. Here, we explore the family’s biological diversity by characterizing the first archaeal and hyperthermophilic GH53 galactanase, and utilize it as a scaffold for engineering enzymes with different product lengths. Results A galactanase gene was identified in the genome of the anaerobic hyperthermophilic archaeon Ignisphaera aggregans, and the isolated catalytic domain expressed and characterized (IaGal). IaGal presents the typical (βα)8 barrel structure of clan GH-A enzymes, with catalytic carboxylates at the end of the 4th and 7th barrel strands. Its activity optimum of at least 95 °C and melting point over 100 °C indicate extreme thermostability, a very advantageous property for industrial applications. If enzyme depletion is reduced, so is the need for re-addition, and thus costs. The main stabilizing features of IaGal compared to other structurally characterized members are π–π and cation–π interactions. The length of the substrate binding site—and thus produced oligosaccharide products—is intermediate compared to previously characterized galactanases. Variants inspired by the structural diversity in the GH53 family were rationally designed to shorten or extend the substrate binding groove, in order to modulate product length. Subsite-deleted variants produced shorter products than IaGal, as do the fungal galactanases inspiring the design. IaGal variants engineered with a longer binding site produced a less expected degradation pattern, though still different from that of wild-type IaGal. All variants remained extremely stable. Conclusions We have characterized in detail the most thermophilic endo-β-1,4-galactanase known to date and successfully engineered it to modify the degradation profile, while maintaining much of its desirable thermostability. This is an important achievement as oligosaccharide products length is an important property for industrial and natural GHs alike.

Tofacitinib (CP-690,550), an approved Janus kinase (JAK) inhibitor, has been proven highly efficacious in treating rheumatoid arthritis (RA). Unfortunately, tofacitinib's clinical application has been limited by adverse side effects that arise from its pan-JAKs inhibition and ubiquitous biodistribution. In this study, we have examined whether a reactive oxygen species (ROS)-activated prodrug design could be applied to tofacitinib to potentially avoid its systemic JAK inhibition and thereby reduce its adverse effects. The prodrug strategy selected is based on ROS-labile 4-methylphenylboronic acid pro-moieties linked to the drugs via a carbamate linkage (prodrug 1) or a direct C–N bond (prodrug 2). Activation under pathophysiological concentrations of H2O2 was investigated and conversion to desired tofacitinib assessed. The most promising prodrug candidate, prodrug 2, was selected in agreement with relevant in vitro physicochemical assay and in silico predicted ADME properties. Selected candidate prodrug 2 showed a remarkable potency window compared to tofacitinib in an in vitro kinase assay against JAK1 and JAK3 kinases. Importantly, prodrug 2 displayed a similar pharmacokinetic profile to tofacitinib after single dose i.p. administration to DBA/1 mice. This study supports the promising applications of ROS-sensitive tofacitinib prodrugs. Their further development and applicability would enhance tofacitinib's therapeutic efficacy, and may provide an opportunity for future development of safer tofacitinib dosing regimens.

The liquid-based Carbo-gel technology enables real-time multimodal image guidance for improved therapeutic precision., Diagnostic imaging often outperforms the surgeon’s ability to identify small structures during therapeutic procedures. Smart soft tissue markers that translate the sensitivity of diagnostic imaging into optimal therapeutic intervention are therefore highly warranted. This paper presents a unique adaptable liquid soft tissue marker system based on functionalized carbohydrates (Carbo-gel). The liquid state of these markers allows for high-precision placement under image guidance using thin needles. Based on step-by-step modifications, the image features and mechanical properties of markers can be optimized to bridge diagnostic imaging and specific therapeutic interventions. The performance of Carbo-gel is demonstrated for markers that (i) have radiographic, magnetic resonance, and ultrasound visibility; (ii) are palpable and visible; and (iii) are localizable by near-infrared fluorescence and radio guidance. The study demonstrates encouraging proof of concept for the liquid marker system as a well-tolerated multimodal imaging marker that can improve image-guided radiotherapy and surgical interventions, including robotic surgery.

Fragment‐based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide‐spread use, the approach still suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp3‐rich fragments is shape diverse and natural‐product‐like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two‐stage workflow of 19F NMR and subsequent 1H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor‐made for 19F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community., We are grateful to DTU Chemistry for an academic excellence PhD scholarship for NST. The NMR Center • DTU and the VillumFoundation are acknowledged for access toNMR spectrometers. CR, HZ, and ES thank the DFG (RA1944/7-1) and the Max Planck Society for support. This work was supported by a grant from the Spanish Ministry ofScience and Innovation (SAF2016-79792R) to AC and DGR. We thank Dr. Kasper Enemark-Rasmussen and Charlie Johansen for expert technical assistance and Anastasia Richter, Mie Larsen, Sanne Møller, Thomas Klevin, Joakim Svensson, Julie Forchhammer, Pernille Christensen, and Ana Laura da Silva for synthesis and analysis of some fragments.

Fragment-based drug discovery (FBDD) has become an established approach for the generation of early lead candidates. However, despite its success and inherent advantages, hit-to-candidate progression for FBDD is not necessarily faster than for the traditional high-throughput screening. Thus, new technology-driven library design strategies have emerged as a means to facilitate more efficient fragment screening and/or subsequent fragment-to-hit chemistry. This minireview will discuss such strategies, which cover the use of labelled fragments for NMR spectroscopy, X-ray crystallographic screening of specialized fragments, covalent linkage for mass spectrometry, dynamic combinatorial chemistry, and fragments optimized for easy elaboration.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes severe joints damage and other extra-articular alterations. Despite the efficacy of low-dose methotrexate (LD-MTX) in RA treatment, adverse effects are the predominant reasons for discontinuation of therapy. As a therapeutic targeting strategy, the presence of increased concentrations of reactive oxygen species (ROS) in the inflammatory environment can serve as the stimulus for prodrug activation in siteselective drug delivery systems. Our group has previously reported novel ROS sensitive prodrugs (1– 3) of MTX and aminopterin (AMT) for site-selective delivery to inflammatory tissue associated with RA, with the aim of reducing side effects in RA therapy. Herein, we investigate the effect and toxicity of the same prodrugs in a rat CIA (collagen-induced arthritis) model of RA. We find that prodrug 1, an arylboronic acid ROS-sensitive MTX-prodrug, displays similar in vivo efficacy as MTX at an equimolar dose, while avoiding adverse effects known to restrict MTX treatment. To further characterize prodrug 1 and its ROS mediated activation, we synthesized compound 4, a negative control lacking the boronic acid moiety. We then investigated the effect of molecules on cell proliferation and cytotoxicity in the presence of the ROS scavenger pyruvate, as well as their stability in buffer and cell media, demonstrating a direct correlation between ROS concentration and the prodrug activity. Moreover, the in vitro ADME properties were investigated, including permeability, rat plasma and microsomal stability.

Background Identification and characterization of key enzymes associated with cell wall biosynthesis and modification is fundamental to gain insights into cell wall dynamics. However, it is a challenge that activity assays of glycosyltransferases are very low throughput and acceptor substrates are generally not available. Results We optimized and validated microscale thermophoresis (MST) to achieve high throughput screening for glycosyltransferase substrates. MST is a powerful method for the quantitative analysis of protein–ligand interactions with low sample consumption. The technique is based on the motion of molecules along local temperature gradients, measured by fluorescence changes. We expressed glycosyltransferases as YFP-fusion proteins in tobacco and optimized the MST method to allow the determination of substrate binding affinity without purification of the target protein from the cell lysate. The application of this MST method to the β-1,4-galactosyltransferase AtGALS1 validated the capability to screen both nucleotide-sugar donor substrates and acceptor substrates. We also expanded the application to members of glycosyltransferase family GT61 in sorghum for substrate screening and function prediction. Conclusions This method is rapid and sensitive to allow determination of both donor and acceptor substrates of glycosyltransferases. MST enables high throughput screening of glycosyltransferases for likely substrates, which will narrow down their in vivo function and help to select candidates for further studies. Additionally, this method gives insight into biochemical mechanism of glycosyltransferase function.

Floral colour change is a widespread phenomenon in angiosperms, but poorly understood from the genetic and chemical point of view. This article investigates this phenomenon in Pleroma raddianum, a Brazilian endemic species whose flowers change from white to purple. To this end, flavonoid compounds and their biosynthetic gene expression were profiled. By using accurate techniques (Ultra Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UPLC-HRMS)), thirty phenolic compounds were quantified. Five key genes of the flavonoid biosynthetic pathway were partially cloned, sequenced, and the mRNA levels were analysed (RT-qPCR) during flower development. Primary metabolism was also investigated by gas chromatography coupled to mass spectrometry (GC-EIMS), where carbohydrates and organic acids were identified. Collectively, the obtained results suggest that the flower colour change in P. raddianum is determined by petunidin and malvidin whose accumulation coincides with the transcriptional upregulation of early and late biosynthetic genes of the flavonoid pathway, mainly CHS and ANS, respectively. An alteration in sugars, organic acids and phenolic co-pigments is observed together with the colour change. Additionally, an increment in the content of Fe3+ ions in the petals, from the pink to purple stage, seemed to influence the saturation of the colour.

A series of novel hydrogen peroxide sensitive prodrugs of methotrexate (MTX) and aminopterin (AMT) were synthesized and evaluated for therapeutic efficacy in mice with collagen induced arthritis (CIA) as a model of chronic rheumatoid arthritis (RA). The prodrug strategy selected is based on ROS-labile 4-methylphenylboronic acid promoieties linked to the drugs via a carbamate linkage or a direct C-N bond. Activation under pathophysiological concentrations of H2O2 proved to be effective, and prodrug candidates were selected in agreement with relevant in vitro physicochemical and pharmacokinetic assays. Selected candidates showed moderate to good solubility, high chemical and enzymatic stability, and therapeutic efficacy comparable to the parent drugs in the CIA model. Importantly, the prodrugs displayed the expected safer toxicity profile and increased therapeutic window compared to MTX and AMT while maintaining a comparable therapeutic efficacy, which is highly encouraging for future use in RA patients.

By using iridium catalysed dehydrogenative decarbonylation, we converted a partly protected cellobioside into a fully protected xylobioside. We demonstrate good yields with two different aromatic ester protecting groups. The resulting xylobioside was directly used as glycosyl donor in further synthesis of a xylooctaose.

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories world-wide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope for only a few of these antibodies has been determined. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision.

Plant cell walls are composed of an intricate network of polysaccharides and proteins that varies during the developmental stages of the cell. This makes it very challenging to address the functions of individual wall components in cells, especially for highly complex glycans. Fortunately, structurally defined oligosaccharides can be used as models for the glycans, to study processes such as cell wall biosynthesis, polysaccharide deposition, protein-carbohydrate interactions, and cell-cell adhesion. Synthetic chemists have focused on preparing such model compounds, as they can be produced in good quantities and with high purity. This review contains an overview of those plant and algal polysaccharides, which have been elucidated to date. The majority of the content is devoted to detailed summaries of the chemical syntheses of oligosaccharide fragments of cellulose, hemicellulose, pectin, and arabinogalactans, as well as glycans unique to algae. Representative synthetic routes within each class are discussed in detail and the progress in carbohydrate chemistry over recent decades is highlighted.

The one-step synthesis and characterization of a series of regioisomerically pure 5-carboxy-seminaphthofluoresceins (5-carboxy-SNAFLs) is reported. The optical properties were determined in aqueous buffer at around biological pH, and highly pH sensitive, large Stokes-shift fluorophores with emission in the deep-red to near-infrared region were identified.

Career situation of first and presenting author Post-doctoral fellow. Introduction The use of methotrexate (MTX) for the treatment of rheumatoid arthritis (RA) is limited by serious adverse effects. Some effects, such as stomatitis and gastric ulcer, can be alleviated by folate supplement. Others are folate-independent, e.g. fibrosis of liver and lung. Moreover, MTX further elevates the micronuclei count, which is generally already increased in RA. In RA, inflamed tissue is characterized by up to 100-fold increased concentrations of reactive oxygen species (ROS, including hydrogen peroxide) compared to healthy tissue. A novel MTX prodrug, ROS101 in development for the treatment of RA, releases MTX at exposure to ROS. This restricts MTX exposure to target tissues with increased ROS levels, e.g. the synovial membrane in RA. Objectives To investigate the effect and toxicity of a targeted MTX prodrug in a rat CIA model of rheumatoid arthritis. Methods ROS101 was tested in a collagen induced arthritis (CIA) model in dark agouti (DA) rats (10 per group) against vehicle and 0.3 mg MTX i.p./day at equimolar doses. Arthritis was induced by s.c. injection of an emulsion of collagen type II in complete Freund’s adjuvant. Treatment was initiated at a mean arthritis severity score of 2/60. Blinded disease evaluation took place 3 times/week from day 10. Bone marrow cells for micronucleus test were obtained from the femur, pictures captured with LARS.4.8 software and analyzed blinded. Individual rat data were included until termination. Results Mean and maximum arthritis severity score was reduced in rats in the ROS101 group compared to vehicle (mean score 6.3±3.1 vs. 14.1±3.3, mean±SEM); (max score 12.6±5.3 vs. 32.3±5.3, mean±SEM, p Conclusions The CIA study in rats indicates that the MTX prodrug ROS101 may be efficacious for the treatment of RA at an equimolar dose compared to MTX, while avoiding adverse effects known to restrict treatment with MTX. Acknowledgements C.A. Hansen, co-founder of ROS Therapeutics. Disclosure of Interest V. Previtali: None declared, N. Woodworth: None declared, M. Hopkins: None declared, J. Petersen Shareholder of: ROS Therapeutics, Consultant for: ROS Therapeutics, I. Ahnfelt-Ronne Shareholder of: ROS Therapeutics, Consultant for: ROS Therapeutics, M. Clausen Shareholder of: ROS Therapeutics.

Compound screening in biological assays and subsequent optimization of hits is indispensable for the development of new molecular research tools and drug candidates. To facilitate such discoveries, the European Research Infrastructure EU-OPENSCREEN was founded recently with the support of its member countries and the European Commission. Its distributed character harnesses complementary knowledge, expertise, and instrumentation in the discipline of chemical biology from 20 European partners, and its open working model ensures that academia and industry can readily access EU-OPENSCREEN’s compound collection, equipment, and generated data. To demonstrate the power of this collaborative approach, this perspective article highlights recent projects from EU-OPENSCREEN partner institutions. These studies yielded (1) 2-aminoquinazolin-4(3H)-ones as potential lead structures for new antimalarial drugs, (2) a novel lipodepsipeptide specifically inducing apoptosis in cells deficient for the pVHL tumor suppressor, (3) small-molecule-based ROCK inhibitors that induce definitive endoderm formation and can potentially be used for regenerative medicine, (4) potential pharmacological chaperones for inborn errors of metabolism and a familiar form of acute myeloid leukemia (AML), and (5) novel tankyrase inhibitors that entered a lead-to-candidate program. Collectively, these findings highlight the benefits of small-molecule screening, the plethora of assay designs, and the close connection between screening and medicinal chemistry within EU-OPENSCREEN. The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: NOR-OPENSCREEN, funded by the Research Council of Norway (RCN) and the K.G. Jebsen Centre for Neuropsychiatric Disorders, for financial support (to A.M.). The Danish Research Infrastructure for Chemical Biology, DK-OPENSCREEN, acknowledges financial support from the Ministry of Higher Education and Science (grant case no. 5072-00019B), the Technical University of Denmark, and the other contributing universities. The Latvian Institute of Organic Synthesis acknowledges the European Regional Development Fund (agreement no. 1.1.1.1/16/A/290) for financial support. L.L. acknowledges the Academy of Finland (grant nos. 287063 and 294085) and the Jane and Aatos Erkko Foundation for funding. EU-OPENSCREEN acknowledges its member and observer states for funding and support. P. Bartunek acknowledges MEYS (LO1220 and LM2015063) for funding. P.G. and H.L. acknowledge funding from the German Federal Ministry of Education and Research. J.L.K. and R.P. acknowledge financial support from the Polish Ministry of Science and Higher Education (KNOW program and POL-OPENSCREEN project number 401086). M.J.V. acknowledges the European Regional Development Fund and the Conselleria de Sanitat Universal i Salut Pública (Generalitat Valenciana, GVA) SI

Members of the CUS family, suspected to be responsible for cutin polymerization, have shown in vitro activity towards sn-2 glyceryl ester (2-MAGs). To assess the selectivity of CUS enzymes towards esters with different headgroups, the CUS1 mediated in vitro polymerization of four monomers of 10,16-dihydroxyhexadecanoic acid was investigated. Our results demonstrated that CUS1 is more selective towards sn-2 glyceryl ester, reinforcing the hypothesis that 2-MAGs are the monomeric precursors of the plant polyester cutin. Additionally, the absence of oxygen atoms in the headgroup negatively impacts the efficiency of polymerization.

Increased levels of reactive oxygen species (ROS) have been associated with numerous pathophysiological conditions including cancer and inflammation and the ROS stimulus constitutes a potential trigger for drug delivery strategies. Over the past decade, a number of ROS-sensitive functionalities have been identified with the purpose of introducing disease-targeting properties into small molecule drugs – a prodrug strategy that offers a promising approach for increasing the selectivity and efficacy of treatments. This review will provide an overview of the ROS-responsive prodrugs developed to date. A discussion on the current progress and limitations is provided along with a reflection on the unanswered questions that need to be addressed in order to advance this novel approach to the clinic.

Byproducts in metalnanoparticle synthesis can interfere with nanomaterial formation and self-assembly, as well as the perceived nanomaterial properties. Such syntheses go through a complicated series of intermediates, making it difficult to predict byproduct chemistry and challenging to determine experimentally. By a combined experimental and theoretical approach, the formation of organic byproducts is mapped out for the synthesis of gold nanoparticles with Good’s buffer 2-(N-morpholino)ethanesulfonic acid. Comprehensive nuclear magnetic resonance studies supported by mass spectrometry, ultraviolet–visible spectroscopy, and density functional theory reveal a number of previously unidentified byproducts formed by oxidation, C–N bond cleavage,and C–C bond formation. A reaction mechanism involving up to four consecutive oxidations is proposed. Oligomeric products with electronic transitions in the visible range are suggested. This approach can be extended broadly and lead to a more informed synthesis designand material characterization.

The synthesis of linear- and (1→6)-branched β-(1→4)-d-galactans, side-chains of the pectic polysaccharide rhamnogalacturonan I is described. The strategy relies on iterative couplings of n-pentenyl disaccharides followed by a late stage glycosylation of a common hexasaccharide core. Reaction with a covalent linker and immobilization on N-hydroxysuccinimide (NHS)-modified glass surfaces allows the generation of carbohydrate microarrays. The glycan arrays enable the study of protein-carbohydrate interactions in a high-throughput fashion, demonstrated herein with binding studies of mAbs and a CBM.

Small-molecule kinase inhibitors (SMKIs), 28 of which are approved by the US Food and Drug Administration (FDA), have been actively pursued as promising targeted therapeutics. Here, we assess the key structural and physicochemical properties, target selectivity and mechanism of function, and therapeutic indications of these approved inhibitors. Our analysis showed that >30% of approved SMKIs have a molecule weight (MW) exceeding 500 and all have a total ring count of between three and five. The assumption that type II inhibitors tend to be more selective than type I inhibitors has been proved to be unreliable. Although previous SMKI research was concentrated on tyrosine kinase inhibitors for cancer treatment, recent progress indicates diversification of SMKI research in terms of new targets, mechanistic types, and therapeutic indications.

Background:Rheumatoid arthritis (RA) is an immune mediated inflammatory disease with autoimmune features, including antibodies to citrullinated proteins and peptides (ACPAs). Several in vitro studies have suggested a pathogenic role of ACPAs in RA. However, in vivo proof of this concept has been hampered by the lack of therapeutic strategies to reduce or deplete ACPA in serum and synovial fluid. Previously, we constructed a chitosan-hyaluronic acid nanoparticle formulation with the ability to use neutrophil recruitment as a delivery mechanism to inflamed joints. Specifically, nanoparticles got phagocytosed and then released to synovial fluid upon death of the short-lived neutrophilsObjectives:We hypothesized that reducing ACPA levels would have a therapeutic effect by blocking cytokine production. In this study, we prepared and tested a series of therapeutic nanoparticles for specific targeting of ACPA in synovial fluid.Methods:Nanoparticles were prepared by the microdroplet method and then decorated with synthetic cyclic citrullinated peptide aptamer PEP2, PEG/hexanoic acid and fluorophore (Cy5.5). Nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC). Nanoparticles were then used in a series of in vitro assays, including cell uptake with flow cytometry (FACS) detection, and in vivo studies including disease activity scores, cytokine measurements and near-infrared imaging.Results:We screened a series of citrullinated peptide epitopes and identified a fibrinogen-derived 21-amino-acid-long citrullinated peptide showing high selectivity toward autoantibodies in RA samples. We incorporated this aptamer in the chitosan-hyaluronic acid nanoparticle formulation previously described. Average nanoparticle size was 230 nm ± 10 nm by DLS and SEM; z potential was -0.0012. Purity by HPLC was over 95%. Attachment efficiency of the aptamer was 92% by HPLC. FACS study showed selective uptake of Cy5.5 labelled aptamer-nanoparticle conjugates by neutrophils in the concentration range 0.5-4 nM. Similar to previous studies,1there was no apparent immunogenicity for this nanoparticle formulation measured by cytokine secretion from human peripheral blood leukocytes. In vivo, over 50% reduction of disease activity was achieved in three weeks treatment using as little as 1 nM drug candidate (dosed every 48 hours) in the collagen-induced (CIA) mouse model of RA (N=30; pConclusion:Overall, we have designed a first-in-class therapeutic nanoparticle drug for specific targeting of anti-citrullinated protein antibodies. The marked effect of this nanoparticle observed in vivo holds promise for targeting ACPAs as a therapeutic option in RA.References:[1]Khatri S, Hansen J, Mendes AC, Chronakis IS, Hung S-C, Mellins ED, Astakhova K. Bioconjug Chem. 2019 Oct 16;30(10):2584–259Disclosure of Interests:Sangita Khatri: None declared, Jonas Hansen: None declared, Mads Hartvig Clausen Shareholder of: iBio Tech ApS, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Shu-Chen Hung: None declared, Elisabeth Mellins: None declared, Kira Astakhova: None declared

Pectin is a major component of primary cell walls and performs a plethora of functions crucial for plant growth, development and plant-defense responses. Despite the importance of pectic polysaccharides their biosynthesis is poorly understood. Several genes have been implicated in pectin biosynthesis by mutant analysis, but biochemical activity has been shown for very few. We used reverse genetics and biochemical analysis to study members of Glycosyltransferase Family 92 (GT92) in Arabidopsis thaliana. Biochemical analysis gave detailed insight into the properties of GALS1 (Galactan synthase 1) and showed galactan synthase activity of GALS2 and GALS3. All proteins are responsible for adding galactose onto existing galactose residues attached to the rhamnogalacturonan-I (RG-I) backbone. Significant GALS activity was observed with galactopentaose as acceptor but longer acceptors are favored. Overexpression of the GALS proteins in Arabidopsis resulted in accumulation of unbranched β-1, 4-galactan. Plants in which all three genes were inactivated had no detectable β-1, 4-galactan, and surprisingly these plants exhibited no obvious developmental phenotypes under standard growth conditions. RG-I in the triple mutants retained branching indicating that the initial Gal substitutions on the RG-I backbone are added by enzymes different from GALS.

Methotrexate (MTX) is the standard of care in the treatment of rheumatoid arthritis (RA), a common autoimmune disease that is characterized by chronic inflammation in the synovial membrane of joints. Unfortunately, MTX suffers from high discontinuation rates due to a large variability in efficacy and, in particular, adverse effects. As inflammation is associated with elevated levels of reactive oxygen species (ROS) like H2O2, we propose to improve treatment through site-selective delivery of MTX to inflammatory tissue by use of a H2O2 sensitive MTX prodrug. To establish proof proof-of-concept, two novel H2O2 sensitive, thiazolidinone-based MTX prodrugs were synthesized and evaluated for this purpose. MTX-γ-thiazolidinone (MTX-γ-TZ) exhibited the most promising properties – good to high chemical and metabolic stability, excellent aqueous solubility, while being activated when subjected to patho-physiological concentrations of H2O2. In vivo, MTX-γ-TZ exhibited comparable efficacy to MTX in a murine collagen type II-induced arthritis (CIA) model while treated mice showed indications of reduced toxicity as their body weight decreased less towards the end of the study, compared to the MTX-treated group

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations, and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side chains of rhamnogalacturonan-I. In Arabidopsis, Galactan Synthase 1 (GALS1) catalyzes the addition of galactose units from UDP-Gal to growing β-1,4-galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP-α-d-Gal and the transfer of an arabinopyranose from UDP-β-l-Arap to galactan chains. The two substrates share a similar structure, but UDP-α-d-Gal is the preferred substrate, with a tenfold higher affinity. Transfer of Arap to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bi-functionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage. This article is protected by copyright. All rights reserved.

The synthesis of two protected tetrasaccharide pentenyl glycosides with diarabinan and digalactan branching related to the pectic polysaccharide rhamnogalacturonan I is reported. The strategy relies on the coupling of N-phenyl trifluoroacetimidate disaccharide donors to a common rhamnosyl acceptor. The resulting trisaccharide thioglycosides were finally coupled to an n-pentenyl galactoside acceptor to access the two protected branched tetrasaccharides.

We report the synthesis of linear and branched (1→4)-d-galactans. Four tetrasaccharides and one pentasaccharide were accessed by adopting a procedure of regioselective ring opening of a 4,6-O-naphthylidene protecting group followed by glycosylation using phenyl thioglycoside donors. The binding of the linear pentasaccharide with galectin-3 is also investigated by the determination of a co-crystal structure. The binding of the (1→4)-linked galactan to Gal-3 highlights the oligosaccharides of pectic galactan, which is abundant in the human diet, as putative Gal-3 ligands.