Ranking Hits From Saturation Transfer Difference Nuclear Magnetic Resonance–Based Fragment Screening

Fragment-based screening is an established route to identify low-molecular-weight molecules to generate high-affinity inhibitors in drug discovery. The affinities of these early hits from fragment screenings require a highly sensitive biophysical screening technique. Saturation transfer difference (STD) nuclear magnetic resonance (NMR) is one of the most popular methods owing to its high sensitivity for low-affinity ligands. It would be highly beneficial if rank-ordering of hits according to their affinity from an initial or counter-screen could be performed—a selection criterion found in the literature. We applied Complete Relaxation and Conformational Exchange Matrix (CORCEMA) theory adapted for saturation transfer (ST) measurements (CORCEMA-ST) calculations to predict STD NMR results from a large set of fragment/receptor pairs to investigate the boundaries under which the assumption holds true that a high STD effect can be applied to select for higher-affinity fragments. Overall, we come to the conclusion that this assumption is invalid

Editorial: Addressing roles for glycans in immunology using chemical biology

Glycoconjugates, macromolecules containing carbohydrates (glycans) conjugated to proteins or lipids, are a diverse class of biopolymers capable of regulating cell-cell interactions. They are present in a high, natural heterogeneity, which originates from the complex mechanisms involved in their biosynthesis. Genetic and environmental factors determine the ensemble of glycans on any particular cell type, in a non-template encoded manner. As a consequence, the cell surface glycan profile provides a tightly-regulated temporal and spatial signature containing crucial biological information. This information is translated into biological functions by glycan binding proteins (GBPs), also called lectins. Importantly, our immune system is modulated by three major GBP families: C-type lectins, galectins, and Siglecs. The abilities of these GBPs to modulate immune cell function is intimately connected to their ability to differentiate ?self? or ?non-self? glycans from our own cells or pathogens, respectively. Hence, GBP?glycan interactions are critical mediators in immune cell homeostasis. Genetic manipulation of glycan processing enzymes has shed light on the roles of glycans in pathologies such as autoimmune diseases and cancer. However, genetic tools such as genomic manipulation and transgenic animal models have shown to be insufficient to fully untangle the roles of GBP-glycan interactions. Accordingly, recent advances in our understanding of GBPs and how they control immune cell function via glycan recognition has been driven by the development of chemical tools.In this Research Topic, we explore recent work illuminating the various roles of glycans and/or GBPs in controlling immune cell function with special emphasis placed on chemical biology approaches that have been instrumental in such efforts. Potential subjects covered may include:? Immunological roles of Glycan-binding proteins? Glycans as immunomodulators? Development of ligands to probe glycan-binding proteins? Chemical biology approaches to modulate glycan-binding proteins and their glycan ligands? Glycans and synthetic derivatives as novel adjuvants? Glycan-based targeted delivery? Intracellular glycosylation in immune cells? Tissue homing of immune cells mediated by glycans? Glycolipid presentation to immune cells? Glycan-based vaccines? Analytical methods for functional characterization of lectin-glycan interactionsFil: Macauley, Matthew S.. University of Alberta; CanadáFil: Rademacher, Christoph. Max Planck Institute of Colloids and Interfaces; AlemaniaFil: Mariño, Karina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Post-translational timing mechanisms of the Drosophila circadian clock

AbstractCircadian clocks allow a temporal coordination and segregation of physiological, metabolic, and behavioural processes as well as their synchronization with the environmental cycles of day and night. Circadian regulation thereby provides a vital advantage, improving an organisms’ adaptation to its environment. The molecular clock can be synchronized with environmental cycles of day and night, but is able to maintain a self-sustained molecular oscillation also in the absence of environmental stimuli. Interlocked transcriptional-translational feedback loops were shown to form the basis of circadian clock function in all phyla from bacteria, fungi, plants, insects to humans. More recently post-translational regulation was identified to be equally important, if not sufficient for molecular clock function and accurate timing of circadian transcription. Here we review recent insights into post-translational timing mechanisms that control the circadian clock, with a particular focus on Drosophila. Analogous to transcriptional feedback regulation, circadian clock function in Drosophila appears to rely on inter-connected post-translational timers. Post-translational regulation of clock proteins illustrates mechanisms that allow a precise temporal control of transcription factors in general and of circadian transcription in particular

Untersuchung von Magnetronsputterpozessen zur Herstellung optischer Präzisionsbeschichtungen

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Glycomimetics for the inhibition and modulation of lectins

Carbohydrates are essential mediators of many processes in health and disease. They regulate self-/ non-self- discrimination, are key elements of cellular communication, cancer, infection and inflammation, and determine protein folding, function and life-times. Moreover, they are integral to the cellular envelope for microorganisms and participate in biofilm formation. These diverse functions of carbohydrates are mediated by carbohydrate-binding proteins, lectins, and the more the knowledge about the biology of these proteins is advancing, the more interfering with carbohydrate recognition becomes a viable option for the development of novel therapeutics. In this respect, small molecules mimicking this recognition process become more and more available either as tools for fostering our basic understanding of glycobiology or as therapeutics. In this review, we outline the general design principles of glycomimetic inhibitors (Section 2). This section is then followed by highlighting three approaches to interfere with lectin function, i.e. with carbohydrate-derived glycomimetics (Section 3.1), novel glycomimetic scaffolds (Section 3.2) and allosteric modulators (Section 3.3). We summarize recent advances in design and application of glycomimetics for various classes of lectins of mammalian, viral and bacterial origin. Besides highlighting design principles in general, we showcase defined cases in which glycomimetics have been advanced to clinical trials or marketed. Additionally, emerging applications of glycomimetics for targeted protein degradation and targeted delivery purposes are reviewed in Section 4

Computational and experimental prediction of human C-type lectin receptor druggability

Mammalian C-type lectin receptors (CTLRS) are involved in many aspects of immune cell regulation such as pathogen recognition, clearance of apoptotic bodies, and lymphocyte homing. Despite a great interest in modulating CTLR recognition of carbohydrates, the number of specific molecular probes is limited. To this end, we predicted the druggability of a panel of 22 CTLRs using DoGSiteScorer. The computed druggability scores of most structures were low, characterizing this family as either challenging or even undruggable. To further explore these findings, we employed a fluorine-based nuclear magnetic resonance screening of fragment mixtures against DC-SIGN, a receptor of pharmacological interest. To our surprise, we found many fragment hits associated with the carbohydrate recognition site (hit rate = 13.5%). A surface plasmon resonance-based follow-up assay confirmed 18 of these fragments (47%) and equilibrium dissociation constants were determined. Encouraged by these findings we expanded our experimental druggability prediction to Langerin and MCL and found medium to high hit rates as well, being 15.7 and 10.0%, respectively. Our results highlight limitations of current in silico approaches to druggability assessment, in particular, with regard to carbohydrate-binding proteins. In sum, our data indicate that small molecule ligands for a larger panel of CTLRs can be developed

Variable selection procedures and efficient suboptimal mask search algorithms in fuzzy inductive reasoning

This paper describes two new suboptimal mask search algorithms for Fuzzy inductive reasoning (FIR), a technique for modelling dynamic systems from observations of their input/output behaviour. Inductive modelling is by its very nature an optimisation problem. Modelling large-scale systems in this fashion involves solving a high-dimensional optimisation problem, a task that invariably carries a high computational cost. Suboptimal search algorithms are therefore important. One of the two proposed algorithms is a new variant of a directed hill-climbing method. The other algorithm is a statistical technique based on spectral coherence functions. The utility of the two techniques is demonstrated by means of an industrial example. A garbage incinerator process is inductively modelled from observations of 20 variable trajectories. Both suboptimal search algorithms lead to similarly good models. Each of the algorithms carries a computational cost that is in the order of a few percent of the cost of solving the complete optimisation problem. Both algorithms can also be used to filter out variables of lesser importance, i.e. they can be used as variable selection tools.Peer Reviewe

Druggable Allosteric Sites in β-Propeller Lectins

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non-carbohydrate drug-like inhibitors are still unavailable. Here, we present a druggable pocket in a β-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing 19F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure–activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol−1 HA−1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecAmediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and D-galactose (D-Gal). Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl D-galactosamine (D-GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-D-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a 6-fold improved binding of D-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA