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High-performance affinity chromatography is limited by its high cost and high pressure. Paper is made up of porous fiber networks and has the properties of low cost, ease of fabrication, and biodegradable. Due to these advantages, herein, we immobilized beta2-adrenoceptor (β2-AR) onto the surface of the polytetrafluoroethylene membrane, a paper-based material, and constructed a G protein-coupled receptor (GPCR)-in-paper chromatographic platform. This platform was characterized by Fourier transform infrared spectroscopy, fluorescence analysis, X-ray photoelectron spectroscopy, and chromatographic studies. These morphological and elemental analysis showed that β2-AR was successfully immobilized on the paper surface. The specific drugs have good retentions on the GPCR-in-paper chromatographic platform. The association constants of salbutamol, terbutaline and bambuterol to β2-AR were calculated to be 2.02 × 104 M−1, 1.15 × 104 M−1, 1.75 × 104 M−1 by adsorption energy distribution, which were in good line with the values from frontal analysis, zonal elution and previous literatures. We demonstrated that the GPCR-in-paper platform was cost-effective, easy to be modified for protein immobilization, and applicable in the receptor-drug interaction analysis. We believe such a platform sheds new light on paper chromatography for receptor-drug interaction analysis and other applications.

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event – A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 2) discusses the recommendations for Hybrid LBA/LCMS and regulatory inputs from major global health authorities. Parts 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event – A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Ligand fishing is a widely used approach for screening active compounds from natural products. Recently, cell membrane (CM) as affinity ligand has been applied in ligand fishing, including cell membrane chromatography (CMC) and CM-coated magnetic bead. However, these methods possess many weaknesses, including complicated preparation processes and time-consuming operation. In this study, cheap and easily available cellulose filter paper (CFP) was selected as carrier of CM and used to fabricate a novel CM-coated CFP (CMCFP) for the first time. The type of CFP was optimized according to the amount of immobilized protein, and the immobilization of CM onto CFP by the insertion and self-fusion process was verified by confocal imaging. The CMCFP exhibited good selectivity and stability and was used for fishing potentially active compounds from extracts of Angelica dahurica. Three potentially active compounds, including bergapten, pabulenol, and imperatorin, were fished out and identified. The traditional Chinese medicine systems pharmacology database and analysis platform was used to build an active compound-target protein network, and accordingly, the gamma-aminobutyric acid receptor subunit alpha-1 (GABRA1) was deduced as potential target of CM for the active compounds of Angelica dahurica. Molecular docking was performed to evaluate the interaction between active compounds and GABRA1, and bergapten was speculated as a new potentially active compound. Compared with other methods, the fishing assay based on CMCFP was more effective, simpler, and cheaper.

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reductive amination to evaluate the multiplexing capacity of luminescence resonance energy transfer (LRET) between UCNPs and quantum dots (QDs). This is the first account of a multiplexed bioassay strategy that demonstrates the principle of use of a single form of UCNP as donor and three different color emitting QDs as acceptors to concurrently determine three analytes. Broad absorbance profiles of green, orange and red QDs that spanned from the first exciton absorption peak to the UV region were in overlap with a blue emission band from UCNPs composed of NaYF4 that was doped with 30% Yb3+, 0.5% Tm3+, allowing for LRET that was stimulated using 980 nm near-infrared radiation. The characteristic narrow and well-defined emission peaks of UCNPs and QDs allowed for the collection of luminescence from each nanoparticle using a band-pass optical filter and an epi-fluorescence microscope. The LRET system was used for the concurrent detection of uidA, Stx1A and tetA gene fragments with selectivity even in serum samples, and reached limits of detection of 26 fmol, 56 fmol and 76 fmol, respectively.

Paper-based point-of-care (POC) devices exhibit the advantages of simplicity, rapidity, trim sizes, and low cost, which are of particular importance for food safety, biological analysis, and medical diagnosis. However, the materials utilized to make paper-based POC rarely produce multiple signals, hampering further applications in diverse situations. Herein, we present an appealing approach, namely Colorimetric-Temperature Dual-Signal Output Sensor (CTDSS), and construct a CTDSS based on coordinative self-assembly biomimetic nanozymes Fe-GMP-L-His CPNs as a proof of concept. These CPNs mimic the structure of horseradish peroxidase (HRP), in which Fe (II) is the center, nucleotide GMP and histidine are chosen as ligands to simulate metal coordination of the pyrrole ring and protein function in HRP, respectively. This strategy allows CPNs to show an excellent peroxidase-like activity, efficiently converting H

The lateral-flow immunoassay (LFA) is a well-established diagnostic technology that has recently seen significant advancements due in part to the rapidly expanding fields of paper diagnostics and paper-fluidics. As LFA-based diagnostics become more complex, it becomes increasingly important to quantitatively determine important parameters during the design and evaluation process. However, current experimental methods for determining these parameters have certain limitations when applied to LFA systems. In this work, we describe our novel methods of combining paper and radioactive measurements to determine nanoprobe molarity, the number of antibodies per nanoprobe, and the forward and reverse rate constants for nanoprobe binding to immobilized target on the LFA test line. Using a model LFA system that detects for the presence of the protein transferrin (Tf), we demonstrate the application of our methods, which involve quantitative experimentation and mathematical modeling. We also compare the results of our rate constant experiments with traditional experiments to demonstrate how our methods more appropriately capture the influence of the LFA environment on the binding interaction. Our novel experimental approaches can therefore more efficiently guide the research process for LFA design, leading to more rapid advancement of the field of paper-based diagnostics.

The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3–7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event – a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC–MS, hybrid ligand-binding assay (LBA)/LC–MS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC–MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC–MS for biotherapeutics and regulatory agencies’ inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event – A Full Immersion Week of Bioanalysis, Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for Small Molecules, Peptides and Small Molecule Biomarkers using LCMS. Part 2 (Biotherapeutics, Biomarkers and Immunogenicity Assays using Hybrid LBA/LCMS and Regulatory Agencies’ Inputs) and Part 3 (LBA: Immunogenicity, Biomarkers and PK Assays) are published in volume 9 of Bioanalysis, issues 23 and 24 (2017), respectively.

Bioactive substances in the Biobio River Basin in Chile were examined by deploying Semi Permeable Membrane Devices (SPMDs) upstream and downstream of 4 pulp mill effluent discharges. Androgenic and estrogenic activity of SPMD extracts were then evaluated using in vitro fish sex steroid receptor binding assays. The results indicated the occurrence of estrogenic type compounds associated with one of the mill discharges. A significant correlation among the presence of these compounds, an increase in gonadosomatic index GSI and induction of hepatic EROD activity of two native fish species was observed. However, no significant presence of mature oocytes in female gonads was detected. Although EROD induction was observed in sites impacted by mill effluents, an increase of its activity occurred towards the downstream areas, suggesting other non-mill sources. More research is needed to understand the environmental changes in context of the new technological improvements in treatment systems to MBBR (Moving Bed Biofilm Reactor) recently implemented by the pulp mill industries.

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid ligand binding assay (LBA)/LCMS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for biotherapeutics, biomarkers and immunogenicity assays using hybrid LBA/LCMS and regulatory agencies' inputs. Part 1 (LCMS for small molecules, peptides and small molecule biomarkers) and Part 3 (LBA: immunogenicity, biomarkers and pharmacokinetic assays) are published in Volume 9 of Bioanalysis, issues 22 and 24 (2017), respectively.

On account of its crucial role in the virus life cycle, SARS-COV-2 NSP13 helicase enzyme was exploited as a promising target to identify a novel potential inhibitor using multi-stage structure-based drug discovery approaches. Firstly, a 3D pharmacophore was generated based on the collected data from a protein-ligand interaction fingerprint (PLIF) study using key interactions between co-crystallised fragments and the NSP13 helicase active site. The ZINC database was screened through the generated 3D-pharmacophore retrieving 13 potential hits. All the retrieved hits exceeded the benchmark score of the co-crystallised fragments at the molecular docking step and the best five-hit compounds were selected for further analysis. Finally, a combination between molecular dynamics simulations and MM-PBSA based binding free energy calculations was conducted on the best hit (compound FWM-1) bound to NSP13 helicase enzyme, which identified FWM-1 as a potential potent NSP13 helicase inhibitor with binding free energy equals −328.6 ± 9.2 kcal/mol., Graphical Abstract

Motivation Intercellular communication (i.e. cell–cell communication) plays an essential role in multicellular organisms coordinating various biological processes. Previous studies discovered that feedback loops between two cell types are a widespread and vital signaling motif regulating development, regeneration and cancer progression. While many computational methods have been developed to predict cell–cell communication based on gene expression datasets, these methods often predict one-directional ligand–receptor interactions from sender to receiver cells and are not suitable to identify feedback loops. Results Here, we describe ligand–receptor loop (LRLoop), a new method for analyzing cell–cell communication based on bi-directional ligand–receptor interactions, where two pairs of ligand–receptor interactions are identified that are responsive to each other and thereby form a closed feedback loop. We first assessed LRLoop using bulk datasets and found our method significantly reduces the false positive rate seen with existing methods. Furthermore, we developed a new strategy to assess the performance of these methods in single-cell datasets. We used the between-tissue interactions as an indicator of potential false-positive prediction and found that LRLoop produced a lower fraction of between-tissue interactions than traditional methods. Finally, we applied LRLoop to the single-cell datasets obtained from retinal development. We discovered many new bi-directional ligand–receptor interactions among individual cell types that potentially control proliferation, neurogenesis and/or cell fate specification. Availability and implementation An R package is available at https://github.com/Pinlyu3/LRLoop. The source code can be found at figshare (https://doi.org/10.6084/m9.figshare.20126138.v1). The datasets can be found at figshare (https://doi.org/10.6084/m9.figshare.20126021.v1). Supplementary information Supplementary data are available at Bioinformatics online.

Novel seven‐membered cyclic imine‐based 1,3‐P,N ligands were obtained by capturing a Beckmann nitrilium ion intermediate generated in situ from cyclohexanone with benzotriazole, and then displacing it by a secondary phosphane under triflic acid promotion. These “cycloiminophosphanes” possess flexible non‐isomerizable tetrahydroazepine rings with a high basicity; this sets them apart from previously reported iminophophanes. The donor strength of the ligands was investigated by using their P‐κ1‐ and P,N‐κ2‐tungsten(0) carbonyl complexes, by determining the IR frequency of the trans‐CO ligands. Complexes with [RhCp*Cl2]2 demonstrated the hemilability of the ligands, giving a dynamic equilibrium of κ1 and κ2 species; treatment with AgOTf gives full conversion to the κ2 complex. The potential for catalysis was shown in the RuII‐catalyzed, solvent‐free hydration of benzonitrile and the RuII‐ and IrI‐catalyzed transfer hydrogenation of cyclohexanone in isopropanol. Finally, to enable access to asymmetric catalysts, chiral cycloiminophosphanes were prepared from l‐menthone, as well as their P,N‐κ2‐RhIII and a P‐κ1‐RuII complexes., Complexes, chirality and catalysts: 1,3‐P,N hybrid ligands are broadly applied in cooperative catalysis, but show limited electronic variation. We report novel analogues with a high N‐basicity, based on 7‐membered cyclic imines. Their transition metal complexes demonstrated their donor strength (W), hemilabile coordination (Rh), and catalytic activity for nitrile hydration (Ru) and transfer hydrogenation (Ru, Ir). Chiral derivatives were also obtained, synthesized from l‐menthone.

FabF (3‐oxoacyl‐[acyl‐carrier‐protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target for new antibiotics. Here, we focus on FabF from P. aeruginosa (PaFabF) as antibiotics against this pathogen are urgently needed. To facilitate exploration of this target we have set up an experimental toolbox consisting of binding assays using bio‐layer interferometry (BLI) as well as saturation transfer difference (STD) and WaterLOGSY NMR in addition to robust conditions for structure determination. The suitability of the toolbox to support structure‐based design of FabF inhibitors was demonstrated through the validation of hits obtained from virtual screening. Screening a library of almost 5 million compounds resulted in 6 compounds for which binding into the malonyl‐binding site of FabF was shown. For one of the hits, the crystal structure in complex with PaFabF was determined. Based on the obtained binding mode, analogues were designed and synthesised, but affinity could not be improved. This work has laid the foundation for structure‐based exploration of PaFabF., Fabulous FabF: FabF is an attractive target for new antibiotics. Herein we describe an experimental toolbox to support structure‐based drug design efforts for FabF from P. aeruginosa. The toolbox contains two types of binding assays (bio‐layer interferometry and NMR assays) and robust conditions for soaking FabF crystals. We demonstrate that this toolbox is fit‐for‐purpose by validating hits obtained from virtual screening.An experimental toolbox for structure‐based hit discovery for P. aeruginosa FabF @BrenkRuth #OpenAccess #antibiotics #SBDD

Vitamin D receptor ligands have potential for the treatment of hyperproliferative diseases and disorders related to the immune system. However, hypercalcemic effects limit their therapeutical uses and call for the development of tissue‐selective new analogs. We have designed and synthesized the first examples of 1α,25‐dihydroxyvitamin D3 analogs bearing an allenic unit attached to the D ring to restrict the side‐chain conformational mobility. The triene system was constructed by a Pd0‐mediated cyclization/Suzuki‐Miyaura cross‐coupling process in the presence of an allenic side chain. The allenic moiety was built through an orthoester‐Claisen rearrangement of a propargylic alcohol. The biological activity and structure of (22S)‐1α,25‐dihydroxy‐17,20‐dien‐24‐homo‐21‐nor‐vitamin D3 bound to binding domain of the vitamin D receptor, provide information concerning side‐chain conformational requirements for biological activity., Looking for side‐chain conformational requirements of vitamin D analogs for selective biological activity, a new active vitamin D analog that has conformationally restricted mobility at the side chain and adopts a similar structure than the natural hormone 1α,25‐dihydroxyvitamin D3 in the binding pocket was designed by docking and synthesized. The highlights of the synthesis include a Pd0‐catalyzed cascade and an orthoester‐Claisen‐rearrangement for the formation of the trienic system and the allenic side‐chain, respectively.

The MT2‐selective melatonin receptor ligand UCM765 (N‐(2‐((3‐methoxyphenyl)(phenyl)amino)ethyl)acetamide), showed interesting sleep inducing, analgesic and anxiolytic properties in rodents, but suffers from low water solubility and modest metabolic stability. To overcome these limitations, different strategies were investigated, including modification of metabolically liable sites, introduction of hydrophilic substituents and design of more basic derivatives. Thermodynamic solubility, microsomal stability and lipophilicity of new compounds were experimentally evaluated, together with their MT1 and MT2 binding affinities. Introduction of a m‐hydroxymethyl substituent on the phenyl ring of UCM765 and replacement of the replacement of the N,N‐diphenyl‐amino scaffold with a N‐methyl‐N‐phenyl‐amino one led to highly soluble compounds with good microsomal stability and receptor binding affinity. Docking studies into the receptor crystal structure provided a rationale for their binding affinity. Pharmacokinetic characterization in rats highlighted higher plasma concentrations for the N‐methyl‐N‐phenyl‐amino derivative, consistent with its improved microsomal stability and makes this compound worthy of consideration for further pharmacological investigation., A taste for stability: Compound 19 is a potent melatonin receptor agonist obtained from the structural optimization of UCM765, an MT2‐selective partial agonist active in vivo, but characterized by poor physicochemical and pharmacokinetic properties. Insertion of a bromine atom on the phenyl‐methylamino scaffold led to a more soluble derivative endowed with higher microsomal stability.

The nonheme iron(IV)‐oxido complex trans‐N3‐[(L1)FeIV=O(Cl)]+, where L1 is a derivative of the tetradentate bispidine 2,4‐di(pyridine‐2‐yl)‐3,7‐diazabicyclo[3.3.1]nonane‐1‐one, is known to have an S=1 electronic ground state and to be an extremely reactive oxidant for oxygen atom transfer (OAT) and hydrogen atom abstraction (HAA) processes. Here we show that, in spite of this ferryl oxidant having the “wrong” spin ground state, it is the most reactive nonheme iron model system known so far and of a similar order of reactivity as nonheme iron enzymes (C−H abstraction of cyclohexane, −90 °C (propionitrile), t 1/2=3.5 sec). Discussed are spectroscopic and kinetic data, supported by a DFT‐based theoretical analysis, which indicate that substrate oxidation is significantly faster than self‐decay processes due to an intramolecular demethylation pathway and formation of an oxido‐bridged diiron(III) intermediate. It is also shown that the iron(III)‐chlorido‐hydroxido/cyclohexyl radical intermediate, resulting from C−H abstraction, selectively produces chlorocyclohexane in a rebound process. However, the life‐time of the intermediate is so long that other reaction channels (known as cage escape) become important, and much of the C−H abstraction therefore is unproductive. In bulk reactions at ambient temperature and at longer time scales, there is formation of significant amounts of oxidation product – selectively of chlorocyclohexane – and it is shown that this originates from oxidation of the oxido‐bridged diiron(III) resting state., The nonheme iron(IV)‐oxido complextrans‐N3‐[(L1)FeIV=O(Cl)]+ is an extremely reactive oxidant for OAT and HAA processes and is the most reactive nonheme iron model system known so far and of similar reactivity as nonheme iron enzymes. Discussed are spectroscopic and kinetic data, supported by a DFT‐based theoretical analysis. In bulk reactions cyclohexane is selectively oxidized to chlorocyclohexane.

A bis(18‐crown‐6) Tröger's base receptor and 4‐substituted hepta‐1,7‐diyl bisammonium salt ligands have been used as a model system to study the interactions between non‐polar side chains of peptides and an aromatic cavity of a protein. NMR titrations and NOESY/ROESY NMR spectroscopy were used to analyze the discrimination of the ligands by the receptor based on the substituent of the ligand, both quantitatively (free binding energies) and qualitatively (conformations). The analysis showed that an all‐anti conformation of the heptane chain was preferred for most of the ligands, both free and when bound to the receptor, and that for all of the receptor‐ligand complexes, the substituent was located inside or partly inside of the aromatic cavity of the receptor. We estimated the free binding energy of a methyl‐ and a phenyl group to an aromatic cavity, via CH‐π, and combined aromatic CH‐π and π‐π interactions to be −1.7 and −3.3 kJ mol−1, respectively. The experimental results were used to assess the accuracy of different computational methods, including molecular mechanics (MM) and density functional theory (DFT) methods, showing that MM was superior., A model system to study interactions between aromatic cavities and non‐polar side chains was developed and studied by different NMR methods, where a weak but evident side‐chain discrimination was observed. The experimental quantitative and qualitative data was used to evaluate different computational methods, with the conclusion that for this system, molecular mechanics gave more accurate results than density functional theory calculations.

Fluorinated groups are essential for drug design, agrochemicals, and materials science. The bis(trifluoromethyl)amino group is an example of a stable group that has a high potential. While the number of molecules containing perfluoroalkyl, perfluoroalkoxy, and other fluorinated groups is steadily increasing, examples with the N(CF3)2 group are rare. One reason is that transfer reagents are scarce and metal‐based storable reagents are unknown. Herein, a set of CuI and AgI bis(trifluoromethyl)amido complexes stabilized by N‐ and P‐donor ligands with unprecedented stability are presented. The complexes are stable solids that can even be manipulated in air for a short time. They are bis(trifluoromethyl)amination reagents as shown by nucleophilic substitution and Sandmeyer reactions. In addition to a series of benzylbis(trifluoromethyl)amines, 2‐bis(trifluoromethyl)amino acetate was obtained, which, upon hydrolysis, gives the fluorinated amino acid N,N‐bis(trifluoromethyl)glycine., Stable storage solutions: Copper(I) and silver(I) complexes with the bis(trifluoromethyl)amido ligand stabilized by pyridine and phosphane ligands have become accessible. These complexes are thermally stable and can be stored under inert conditions; in contrast to the behavior of other metal salts such as M{N(CF3)2} (M=Cu, Ag). The complexes were found to be convenient reagents for nucleophilic substitution and Sandmeyer reactions, thereby providing access to a series of organic substrates with the bis(trifluoromethyl)amino group.

Dengue is a tropical disease caused by the dengue virus (DENV), with an estimate of 300 million new cases every year. Due to the limited vaccine efficiency and absence of effective antiviral treatment, new drug candidates are urgently needed. DENV NS3‐NS2B protease complex is essential for viral post‐translational processing and maturation, and this enzyme has been extensively studied as a relevant drug target. Crystal structures often underestimate NS3‐NS2B flexibility, whereas they can adopt different conformational states depending on the bound substrate. We conducted molecular dynamics simulations (∼30 μs) with a non‐ and covalently bound inhibitor to understand the conformational changes in the DENV‐3 NS3‐NS2B complex. Our results show that the open‐closing movement of the protease exposes multiple druggable subpockets that can be investigated in later drug discovery efforts., The open/closing movement of the NS3‐NS2B protease exposes multiple druggable subpockets that can be investigated in later drug discovery efforts. Considering the conformational shift is key for further drug discovery, since induced‐fit docking would be only relevant for fine tuning.

The syntheses of bis(triazolium)carbazole precursors and their corresponding coinage metal (Au, Ag) complexes are reported. For alkylated triazolium salts, di‐ or tetranuclear complexes with bridging ligands were isolated, while the bis(aryl) analogue afforded a bis(carbene) AuI‐CNC pincer complex suitable for oxidation to the redox‐stable [AuIII(CNC)Cl]+ cation. Although the ligand salt and the [AuIII(CNC)Cl]+ complex were both notably cytotoxic toward the breast cancer cell line MDA‐MB‐231, the AuIII complex was somewhat more selective. Electrophoresis, viscometry, UV‐vis, CD and LD spectroscopy suggest the cytotoxic [AuIII(CNC)Cl]+ complex behaves as a partial DNA intercalator. In silico screening indicated that the [AuIII(CNC)Cl]+ complex can target DNA three‐way junctions with good specificity, several other regular B‐DNA forms, and Z‐DNA. Multiple hydrophobic π‐type interactions involving T and A bases appear to be important for B‐form DNA binding, while phosphate O⋅⋅⋅Au interactions evidently underpin Z‐DNA binding. The CNC ligand effectively stabilizes the AuIII ion, preventing reduction in the presence of glutathione. Both the redox stability and DNA affinity of the hit compound might be key factors underpinning its cytotoxicity in vitro., Towards new AuIII anticancer agents. Herein a redox‐stabilised DNA‐targeting AuIII bis(carbene) pincer complex with significant cytotoxicity is reported that is likely underpinned by partial DNA intercalation. In silico studies indicate that the complex can also target unusual DNA forms such as 3‐way junctions and Z‐DNA. The structures of several multinuclear AgI complexes discovered en route to the target AuIII complex were also elucidated.

The 9‐ and 12‐dimethylaminophenyl‐substituted berberine derivatives 3 a and 3 b were readily synthesized by Suzuki‐Miyaura reactions and shown to be useful fluorescent probes for the optical detection of quadruplex DNA (G4‐DNA). Their association with the nucleic acids was investigated by spectrometric titrations, CD and LD spectroscopy, and with DNA‐melting analysis. Both ligands bind to duplex DNA by intercalation and to G4‐DNA by terminal π stacking. At neutral conditions, they bind with higher affinity (K b=105−106 M−1) to representative quadruplex forming oligonucleotides 22AG, c‐myc, c‐kit, and a2, than to duplex calf thymus (ct) DNA (K b=5‐7×104 M−1). At pH 5, however, the affinity of 3 a towards G4‐DNA 22AG is higher (K b=1.2×106 M−1), whereas the binding constant towards ct DNA is lower (K b=3.9×103 M−1) than under neutral conditions. Notably, the association of the ligand with DNA results in characteristic changes of the absorption and emission properties under specific conditions, which may be used for optical DNA detection. Other than the parent berberine, the ligands do not show a noticeable increase of their very low intrinsic emission intensity upon association with DNA at neutral conditions. In contrast, a fluorescence light‐up effect was observed upon association to duplex (Φ fl=0.01) and quadruplex DNA (Φ fl=0.04) at pH 5. This fluorimetric response to G4‐DNA association in combination with the distinct, red‐shifted absorption under these conditions provides a simple and conclusive optical detection of G4‐DNA at lower pH., The very weak fluorescence of 4‐dimethylaminophenyl‐substituted berberines only increases significantly if both their conformational flexibility is strongly restricted, i. e. in viscous medium or when bound to quadruplex DNA, and the amino group is protonated at low pH. This requirement of a dual stimulation to cause a light‐up effect enables the spectrometric detection of quadruplex DNA at pH 5.

A facile imide coupling strategy for the one‐step preparation of G‐quadruplex ligands with varied core chemistries is described. The G‐quadruplex stabilization of a library of nine compounds was examined using FRET melting experiments, and CD, UV‐Vis, fluorescence and NMR titrations, identifying several compounds that were capable of stabilizing G‐quadruplex DNA with interesting selectivity profiles. The best G4 ligand was identified as compound 3, which was based on a perylene scaffold and exhibited 40‐fold selectivity for a telomeric G‐quadruplex over duplex DNA. Surprisingly, a tetra‐substituted flexible core, compound 11, also exhibited selective stabilization of G4 DNA over duplex DNA. The anticancer and antiparasitic activity of the library was also examined, with the lead compound 3 exhibiting nanomolar inhibition of Trypanosoma brucei with 78‐fold selectivity over MRC5 cells. The cellular localization of this compound was also studied via fluorescence microscopy. We found that uptake was time dependant, with localization outside the nucleus and kinetoplast that could be due to strong fluorescence quenching in the presence of small amounts of DNA., The rigid rules of G‐quadruplex stabilization: A library of amphiphiles has been synthesized in one step, using a simple imide condensation reaction. The G‐quadruplex stabilization, anti‐cancer and anti‐parasitic activity of the library has been investigated, uncovering the important factors that govern G‐quadruplex binding and selectivity, yielding a promising new core‐motif and a compound with potent trypanosomacidal activity.

The reaction of UO2(OAc)2 ⋅ 2H2O with the biologically inspired ligand 2‐salicylidene glucosamine (H2 L1) results in the formation of the anionic trinuclear uranyl complex [(UO2)3(μ3‐O)(L1)3]2− (1 2−), which was isolated in good yield as its Cs‐salt, [Cs]2 1. Recrystallization of [Cs]2 1 in the presence of 18‐crown‐6 led to formation of a neutral ion pair of type [M(18‐crown‐6)]2 1, which was also obtained for the alkali metal ions Rb+ and K+ (M=Cs, Rb, K). The related ligand, 2‐(2‐hydroxy‐1‐naphthylidene) glucosamine (H2 L2) in a similar procedure with Cs+ gave the corresponding complex [Cs(18‐crown‐6)]2[(UO2)3(μ3‐O)(L2)3 ([Cs(18‐crown‐6)]2 2). From X‐ray investigations, the [(UO2)3O(Ln)3]2− anion (n=1, 2) in each complex is a discrete trinuclear uranyl species that coordinates to the alkali metal ion via three uranyl oxygen atoms. The coordination behavior of H2 L1 and H2 L2 towards UO2 2+ was investigated by NMR, UV/Vis spectroscopy and mass spectrometry, revealing the in situ formation of the 1 2− and 2 2−dianions in solution., Investigation of the coordination behavior of salicylidene glucosamine (H2 L1) towards uranyl ions (UO2 2+) revealed the exclusive formation of a trinuclear anionic uranyl complex [(UO2)3(μ3‐O)(L1)3]2− (1 2−), which was isolated by precipitation with the alkali metals ions Cs+, Rb+, and K+. Recrystallization of the Cs salt in the presence of 18‐crown‐6 in DMF led to the formation of the solvated ion pair [Cs(18‐crown‐6)]2 1, in which coordination of the Cs+ ion by three uranyl oxygen atoms was observe.

The Cd(II)‐, Pb(II)‐, Ni(II)‐ and Zn(II)‐complexes of small terminally protected peptides containing CXXX, XXXC, XCCX, CXnC (n=1–3) sequences have been studied with potentiometric, UV/Vis and CD spectroscopic techniques. The cysteine thiolate group is the primary binding site for all studied metal ions, but the presence of a histidyl or aspartyl side chain in the molecule contributes to the stability of the complexes. For two‐cysteine containing peptides the (S−,S−) coordinated species are formed in the physiological pH range and the stability increases in the Ni(II), The systematic study of small cysteine containing peptides reveals the outstanding Pb(II)‐ and Cd(II)‐binding ability of peptides with CXnC (n=0–3) sequence. This is well demonstrated by pM values calculated for different metal‐peptide systems at pH 7 (cL=0.010 mM, cM=0.001 mM).

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug‐like compounds was screened in a biosensor‐based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K D=42 and 84 μM, respectively). Co‐crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C‐terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3–HSP90 binding was confirmed (K D=13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action., From a distance: SMYD3 lysine methyltransferase is an epigenetic enzyme with multiple cellular functions and the ability to recognize broad range of substrates from histones to cytosolic proteins. An SPR biosensors‐based biophysical strategy for screening SMYD3 ligands was developed and revealed an allosteric binding site. Subsequent crystallographic studies provided a structural description of the novel binding site of SMYD3.

Three [1,3‐diethyl‐4‐(p‐methoxyphenyl)‐5‐(3,4,5‐trimethoxyphenyl)imidazol‐2‐ylidene](L)gold(I) complexes, 4 a (L=Cl), 5 a (L=PPh3), and 6 a (L=same N‐heterocyclic carbene (NHC)), and their fluorescent [4‐(anthracen‐9‐yl)‐1,3‐diethyl‐5‐phenylimidazol‐2‐ylidene](L)gold(I) analogues, 4 b, 5 b, and 6 b, respectively, were studied for their localisation and effects in cancer cells. Despite their identical NHC ligands, the last three accumulated in different compartments of melanoma cells, namely, the nucleus (4 b), mitochondria (5 b), or lysosomes (6 b). Ligand L was also more decisive for the site of accumulation than the NHC ligand because the couples 4 a/4 b, 5 a/5 b, and 6 a/6 b, carrying different NHC ligands, afforded similar results in cytotoxicity tests, and tests on targets typically found at their sites of accumulation, such as DNA in nuclei, reactive oxygen species and thioredoxin reductase in mitochondria, and lysosomal membranes. Regardless of the site of accumulation, cancer cell apoptosis was eventually induced. The concept of guiding a bioactive complex fragment to a particular subcellular target by secondary ligand L could reduce unwanted side effects., Striking gold: Through the choice of their secondary ligand L, a series of [4,5‐diarylimidazol‐2‐ylidene](L)gold(I) complexes targeted different cancer cell organelles (nuclei, mitochondria or lysosomes) where they interfered beneficially with molecular targets, such as DNA, thioredoxin reductase, or lysosomal membranes, to elicit p53‐independent apoptosis. Such guided metallodrugs will help to minimise unwanted side effects by addressing limited arrays of targets.

In lead optimization, protein crystallography is an indispensable tool to analyze drug binding. Binding modes and non‐covalent interaction inventories are essential to design follow‐up synthesis candidates. Two protocols are commonly applied to produce protein–ligand complexes: cocrystallization and soaking. Because of its time and cost effectiveness, soaking is the more popular method. Taking eight ligand hinge binders of protein kinase A, we demonstrate that cocrystallization is superior. Particularly for flexible proteins, such as kinases, and larger ligands cocrystallization captures more reliable the correct binding pose and induced protein adaptations. The geometrical discrepancies between soaking and cocrystallization appear smaller for fragment‐sized ligands. For larger flexible ligands that trigger conformational changes of the protein, soaking can be misleading and underestimates the number of possible polar interactions due to inadequate, highly impaired positions of protein amino‐acid side and main chain atoms. Thus, if applicable cocrystallization should be the gold standard to study protein–ligand complexes., Lead optimization needs information about binding modes, best from protein crystallography. Cocrystallization and soaking are two protocols to obtain diffracting crystals of protein–ligand complexes. The latter is more popular due to its time and cost effectiveness, but cocrystallization captures the correct binding pose more reliably, particularly of larger and more flexible ligands, as our comparative analysis shows. For fragment‐sized ligands the differences appear negligible.

The conversion of the 1 : 1‐complex of Cisplatin with 1‐methyluracil (1MeUH), cis‐[Pt(NH3)2(1MeU‐N3)Cl] (1 a) to the aqua species cis‐[Pt(NH3)2(1MeU‐N3)(OH2)]+ (1 b), achieved by reaction of 1 a with AgNO3 in water, affords a mixture of compounds, the composition of which strongly depends on sample history. The complexity stems from variations in condensation patterns and partial loss of NH3 ligands. In dilute aqueous solution, 1 a, and dinuclear compounds cis‐[(NH3)2(1MeU‐N3)Pt(μ‐OH)Pt(1MeU‐N3)(NH3)2]+(3) as well as head‐tail cis‐[Pt2(NH3)4(μ‐1MeU‐N3,O4)2]2+ (4) represent the major components. In addition, there are numerous other species present in minor quantities, which differ in metal nuclearity, stoichiometry, stereoisomerism, and Pt oxidation state, as revealed by a combination of 1H NMR and ESI‐MS spectroscopy. Their composition appears not to be the consequence of a unique and repeating coordination pattern of the 1MeU ligand in oligomers but rather the coexistence of distinctly different condensation patterns, which include μ‐OH, μ‐1MeU, and μ‐NH2 bridging and combinations thereof. Consequently, the products obtained should, in total, be defined as a heterogeneous mixture rather than a mixture of oligomers of different sizes. In addition, a N2 complex, [Pt(NH3)(1MeU)(N2)]+ appears to be formed in gas phase during the ESI‐MS experiment. In the presence of Na+ ions, multimers n of 1 a with n=2, 3, 4 are formed that represent analogues of non‐metalated uracil quartets found in tetrastranded RNA., Multiple condensation reactions of mononuclear 1‐methyl uracilato (1MeU) complexes of Cisplatin, and numerous side reactions, lead to a heterogeneous mixture of products, as shown by 1H‐NMR and ESI‐MS

SAM-I riboswitches regulate gene expression through transcription termination upon binding a S-adenosyl-L-methionine (SAM) ligand. In previous work, we characterized the conformational energy landscape of the full-length Bacillus subtilis yitJ SAM-I riboswitch as a function of Mg2+ and SAM ligand concentrations. Here, we have extended this work with measurements on a structurally similar ligand, S-adenosyl-l-homocysteine (SAH), which has, however, a much lower binding affinity. Using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling (HMM) analysis, we identified major conformations and determined their fractional populations and dynamics. At high Mg2+ concentration, FRET analysis yielded four distinct conformations, which we assigned to two terminator and two antiterminator states. In the same solvent, but with SAM added at saturating concentrations, four states persisted, although their populations, lifetimes and interconversion dynamics changed. In the presence of SAH instead of SAM, HMM revealed again four well-populated states and, in addition, a weakly populated ‘hub’ state that appears to mediate conformational transitions between three of the other states. Our data show pronounced and specific effects of the SAM and SAH ligands on the RNA conformational energy landscape. Interestingly, both SAM and SAH shifted the fractional populations toward terminator folds, but only gradually, so the effect cannot explain the switching action. Instead, we propose that the noticeably accelerated dynamics of interconversion between terminator and antiterminator states upon SAM binding may be essential for control of transcription.

Object Find potential therapeutic targets of triple-negative breast cancer (TNBC) patients by bioinformatics. Screen ideal natural ligand that can bind with the potential target and inhibit it by using molecular biology. Methods Bioinformatics and molecular biology were combined to analyze potential therapeutic targets. Differential expression analysis identified the differentially expressed genes (DEGs) between TNBC tissues and non-TNBC tissues. The functional enrichment analyses of DEGs shown the important gene ontology (GO) terms and pathways of TNBC. Protein-protein interaction (PPI) network construction screened 20 hub genes, while Kaplan website was used to analyze the relationship between the survival curve and expression of hub genes. Then Discovery Studio 4.5 screened ideal natural inhibitors of the potential therapeutic target by LibDock, ADME, toxicity prediction, CDOCKER and molecular dynamic simulation. Results 1,212 and 353 DEGs were respectively found between TNBC tissues and non-TNBC tissues, including 88 up-regulated and 141 down-regulated DEGs in both databases. 20 hub genes were screened, and the higher expression of CDC20 was associated with a poor prognosis. Therefore, we chose CDC20 as the potential therapeutic target. 7,416 natural ligands were conducted to bind firmly with CDC20, and among these ligands, ZINC000004098930 was regarded as the potential ideal ligand, owing to its non-hepatotoxicity, more solubility level and less carcinogenicity than the reference drug, apcin. The ZINC000004098930-CDC20 could exist stably in natural environment. Conclusion 20 genes were regarded as hub genes of TNBC and most of them were relevant to the survival curve of breast cancer patients, especially CDC20. ZINC000004098930 was chosen as the ideal natural ligand that can targeted and inhibited CDC20, which may give great contribution to TNBC targeted treatment.

Macrophages rapidly undergo glycolytic reprogramming in response to macroautophagy/autophagy, inflammasome activation and pyroptosis for the clearance of bacteria. Identification the key molecules involved in these three events will provide critical potential therapeutic applications. Upon S. typhimurium infection, FLT4/VEGFR3 and its ligand VEGFC were inducibly expressed in macrophages, and FLT4 signaling inhibited CASP1 (caspase 1)-dependent inflammasome activation and pyroptosis but enhanced MAP1LC3/LC3 activation for elimination of the bacteria. Consistently, FLT4 mutants lacking the extracellular ligand-binding domain increased production of the proinflammatory metabolites such as succinate and lactate, and reduced antimicrobial metabolites including citrate and NAD(P)H in macrophages and liver upon infection. Mechanistically, FLT4 recruited AMP-activated protein kinase (AMPK) and phosphorylated Y247 and Y441/442 in the PRKAA/alpha subunit for AMPK activation. The AMPK agonist AICAR could rescue glycolytic reprogramming and inflammasome activation in macrophages expressing the mutant FLT4, which has potential translational application in patients carrying Flt4 mutations to prevent recurrent infections. Collectively, we have elucidated that the FLT4-AMPK module in macrophages coordinates glycolytic reprogramming, autophagy, inflammasome activation and pyroptosis to eliminate invading bacteria. Abbreviations: 3-MA: 3-methyladenine; AICAR: 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; BMDM: bone marrow-derived macrophage; CASP1: caspase 1; CFUs: colony-forming units; FLT4/VEGFR3: FMS-like tyrosine kinase 4; GFP: green fluorescent protein; LDH: lactate dehydrogenase; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PEM: peritoneal exudate macrophage; PRKAA1/AMPKα1: protein kinase, AMP-activated, alpha 1 catalytic subunit; PYCARD/ASC: PYD and CARD domain containing; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TLR4: toll-like receptor 4; ULK1: unc-51 like autophagy activating kinase 1; VEGFC: vascular endothelial growth factor C; WT: wild type

The protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post‐translational N‐glycosylation of Asn31 of MOG seems to play a key role in modulating the immune response towards myelin. This is mediated by the interaction of Lewis‐type glycan structures in the N‐glycan of MOG with the DC‐SIGN receptor on dendritic cells (DCs). Here, we report the synthesis of an unnatural Lewis X (LeX)‐containing Fmoc‐SPPS‐compatible asparagine building block (SPPS=solid‐phase peptide synthesis), as well as asparagine building blocks containing two LeX‐derived oligosaccharides: LacNAc and Fucα1‐3GlcNAc. These building blocks were used for the glycosylation of the immunodominant portion of MOG (MOG31‐55) and analyzed with respect to their ability to bind to DC‐SIGN in different biological setups, as well as their ability to inhibit the citrullination‐induced aggregation of MOG31‐55. Finally, a cytokine secretion assay was carried out on human monocyte‐derived DCs, which showed the ability of the neoglycopeptide decorated with a single LeX to alter the balance of pro‐ and anti‐inflammatory cytokines, inducing a tolerogenic response., Immunomodulation. A novel asparagine solid‐phase peptide synthesis building block harboring a Lewis X type DC‐SIGN ligand has been synthesized (see scheme) and incorporated into the immunodominant portion of myelin oligodendrocyte glycoprotein (MOG31‐55). This glycopeptide was evaluated for its ability to inhibit citrullination‐induced aggregation of MOG35‐55, to bind to DC‐SIGN in vitro and for its immunomodulatory effects on human dendritic cells, with the Lewis X yielding a tolerogenic response.

10 p.-6 fig.-3 tb., Functional pairing between cellular glyco-con-ju-gates and tissue lectins like galectins has wide (patho)physio-logical significance. Their study is facilitated by non-hydrolyzable deri-va-tives of the natural O-glycans, such as S- and Se-glycosides. The latter en-able extensive analyses by specific 77 Se NMR spectroscopy, but still remain under-explored. By the example of seleno--digalac-to-side (SeDG) and the human galectins 1 and 3, we evaluate diverse 77 Se NMR detec-tion methods and pro-pose selec-tive 1 H, 77 Se hetero-nuclear Hartmann-Hahn transfer for efficient use in com-peti-tive NMR screening against a seleno-glycoside spy ligand. By fluores-cence aniso-tropy, circular dichroism, and isothermal titra-tion calori-metry (ITC) we show that affinity and thermodynamics of SeDG bind-ing by galectins are similar to thiodi-galac-toside (TDG) and N-acetyl-lactos-amine (LacNAc), con-firm-ing that Se substitution has no major impact. ITC data in D 2 O vs. H 2 O are similar for TDG and LacNAc binding by both galectins, but a solvent effect, indicating solvent rearrangement at the binding site, is hinted for SeDG and clearly observed for LacNAc dimers with extend-ed chain length., We are much indebted to L. Szilágyi (University of Debrecen,Hungary) for kindly providing selenoglycosides for this study. We are grateful for inspiring discussions to Drs. B. Friday, A. Leddoz and A. W. L. Nose as well as for generous funding from the Spanish Ministry of Economy and Competitiveness (grant BFU2016-77835-R; to A.R.), the Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Elkartek BG2019; to T.D.), the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644; to T.D.), the Science Foundation of Ireland (grants 13/IA/1959 and 16/RC/3889;to S.O.), National Institute of Health (NIH) grant CA242351 to M.Cudic, and the COST Action CA 18103 (InnoGly; to H.-J.G.).

The formation of a G‐quadruplex motif in the promoter region of the c‐MYC protooncogene prevents its expression. Accordingly, G‐quadruplex stabilization by a suitable ligand may be a viable approach for anticancer therapy. In our study, we used the 4‐(4‐methylpiperazin‐1‐yl)aniline molecule, previously identified as a fragment library screen hit, as a template for the SAR‐guided design of a new small library of clickable fragments and subjected them to click reactions, including kinetic target‐guided synthesis in the presence of a G‐quadruplex forming oligonucleotide Pu24. We tested the clickable fragments and products of click reactions for their G‐quadruplex stabilizing activity and determined their mode of binding to the c‐MYC G‐quadruplex by NMR spectroscopy. The enhanced stabilizing potency of click products in biology assays (FRET, Polymerase extension assay) matched the increased yields of in situ click reactions. In conclusion, we identified the newly synthesized click products of bis‐amino derivatives of 4‐(4‐methylpiperazin‐1‐yl)aniline as potent stabilizers of c‐MYC G‐quadruplex, and their further evolution may lead to the development of an efficient tool for cancer treatment., Stabilization of G‐quadruplexes formed in the c‐MYC promoter region that would prevent expression of this oncogene may be exploited for anticancer therapy. The fragment based approach has been combined with targed‐guided systhesis for the development of small‐molecule G‐quadruplex stabilizers potentially amenable to further therapeutic evolution.

A parallel quadruplex derived from the Myc promoter sequence was extended by a stem‐loop duplex at either its 5′‐ or 3′‐terminus to mimic a quadruplex–duplex (Q–D) junction as a potential genomic target. High‐resolution structures of the hybrids demonstrate continuous stacking of the duplex on the quadruplex core without significant perturbations. An indoloquinoline ligand carrying an aminoalkyl side chain was shown to bind the Q–D hybrids with a very high affinity in the order K a≈107 m −1 irrespective of the duplex location at the quadruplex 3′‐ or 5′‐end. NMR chemical shift perturbations identified the tetrad face of the Q–D junction as specific binding site for the ligand. However, calorimetric analyses revealed significant differences in the thermodynamic profiles upon binding to hybrids with either a duplex extension at the quadruplex 3′‐ or 5′‐terminus. A large enthalpic gain and considerable hydrophobic effects are accompanied by the binding of one ligand to the 3′‐Q–D junction, whereas non‐hydrophobic entropic contributions favor binding with formation of a 2:1 ligand‐quadruplex complex in case of the 5′‐Q–D hybrid., An indoloquinoline‐based G4 ligand binds with very high affinity at the quadruplex–duplex junction of a parallel G‐quadruplex featuring a stem‐loop duplex extension. Thermodynamic profiles and modes of binding differ for hybrids with duplex extensions at either the 5′‐ or 3′‐face of the G‐quadruplex.

Abstract: The BCL‐2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL‐2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non‐antibody‐binding proteins based on a conserved scaffold) to identify ligands for MCL‐1, BCL‐xL, BCL‐2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL‐2 protein. For anti‐apoptotic targets BCL‐xL and MCL‐1, competitive inhibition of their canonical protein‐protein interactions is demonstrated. Co‐crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug‐bound‐like conformation. These proof‐of‐concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL‐2 family modulators and more generally other protein‐protein interaction inhibitors., Affimer reagents (non‐antibody binding proteins) are identified that bind selectively to the anti‐apoptotic proteins BCL‐xL and MCL‐1. Crystallographic studies reveal an unprecedented mode of molecular recognition in which flexible loops from the Affimer dock into the BH3 binding cleft as opposed to a canonical α‐helix. Furthermore the Affimers induce changes in the target proteins towards desirable drug‐bound‐like conformations.

A workflow to expand crystallographic fragment hits to higher affinity compounds using readily available analogs is described and successfully applied., In recent years, crystallographic fragment screening has matured into an almost routine experiment at several modern synchrotron sites. The hits of the screening experiment, i.e. small molecules or fragments binding to the target protein, are revealed along with their 3D structural information. Therefore, they can serve as useful starting points for further structure-based hit-to-lead development. However, the progression of fragment hits to tool compounds or even leads is often hampered by a lack of chemical feasibility. As an attractive alternative, compound analogs that embed the fragment hit structurally may be obtained from commercial catalogs. Here, a workflow is reported based on filtering and assessing such potential follow-up compounds by template docking. This means that the crystallographic binding pose was integrated into the docking calculations as a central starting parameter. Subsequently, the candidates are scored on their interactions within the binding pocket. In an initial proof-of-concept study using five starting fragments known to bind to the aspartic protease endothiapepsin, 28 follow-up compounds were selected using the designed workflow and their binding was assessed by crystallography. Ten of these compounds bound to the active site and five of them showed significantly increased affinity in isothermal titration calorimetry of up to single-digit micromolar affinity. Taken together, this strategy is capable of efficiently evolving the initial fragment hits without major synthesis efforts and with full control by X-ray crystallography.

Understanding protein–ligand interactions in a cellular context is an important goal in molecular biology and biochemistry, and particularly for drug development. Investigators must demonstrate that drugs penetrate cells and specifically bind their targets. Towards that end, we present a native mass spectrometry (MS)‐based method for analyzing drug uptake and target engagement in eukaryotic cells. This method is based on our previously introduced direct‐MS method for rapid analysis of proteins directly from crude samples. Here, direct‐MS enables label‐free studies of protein–drug binding in human cells and is used to determine binding affinities of lead compounds in crude samples. We anticipate that this method will enable the application of native MS to a range of problems where cellular context is important, including protein–protein interactions, drug uptake and binding, and characterization of therapeutic proteins., We report a native mass spectrometry (MS)‐based method for analyzing drug uptake and target engagement in eukaryotic cells, based on our direct‐MS method for rapid analysis of proteins directly from crude samples. We demonstrate label‐free analysis of protein–drug binding in human cells and measurement of the binding affinities of lead compounds in crude samples.

We designed a straightforward synthetic route towards a full‐fledged family of π‐extended helicenes: superhelicenes. They have two hexa‐peri‐hexabenzocoronenes (HBCs) in common that are connected via a central five‐membered ring. By means of structurally altering this 5‐membered ring, we realized a versatile library of molecular building blocks. Not only the superhelicene structure, but also their features are tuned with ease. In‐depth physico‐chemical characterizations served as a proof of concept thereof. The superhelicene enantiomers were separated, their circular dichroism was measured in preliminary studies and concluded with an enantiomeric assignment. Our work was rounded‐off by crystal structure analyses. Mixed stacks of M‐ and P‐isomers led to twisted molecular wires. Using such stacks, charge‐carrier mobilities were calculated, giving reason to expect outstanding hole transporting properties., We present the synthesis and in‐depth physico‐chemical characterization of a family of π‐extended helicenes that consist of HBCs fused via different five‐membered rings. Despite being only a small part of the compounds, the nature of the five‐membered rings has a huge impact on the properties of the whole molecule. We show the enantiomeric separation and circular dichroism properties together with X‐ray analysis and charge transport calculations.

Immunogenic cell death (ICD) offers a method of stimulating the immune system to attack and remove cancer cells. We report a copper(II) complex containing a Schiff base ligand and a polypyridyl ligand, 4, capable of inducing ICD in breast cancer stem cells (CSCs). Complex 4 kills both bulk breast cancer cells and breast CSCs at sub‐micromolar concentrations. Notably, 4 exhibits greater potency (one order of magnitude) towards breast CSCs than salinomycin (an established breast CSC‐potent agent) and cisplatin (a clinically approved anticancer drug). Epithelial spheroid studies show that 4 is able to selectively inhibit breast CSC‐enriched HMLER‐shEcad spheroid formation and viability over non‐tumorigenic breast MCF10 A spheroids. Mechanistic studies show that 4 operates as a Type II ICD inducer. Specifically, 4 readily enters the endoplasmic reticulum (ER) of breast CSCs, elevates intracellular reactive oxygen species (ROS) levels, induces ER stress, evokes damage‐associated molecular patterns (DAMPs), and promotes breast CSC phagocytosis by macrophages. As far as we are aware, 4 is the first metal complex to induce ICD in breast CSCs and promote their engulfment by immune cells., Find me, eat me: Cancer stem cells (CSCs) are heavily linked to patient relapse, so targeting them could improve treatment. One method of inducing a tumour‐targeting immune response is immunogenic cell death (ICD), in which dying cancer cells stimulate tumour‐associated immune cells to seek and remove them. Here we report the first metal complex to induce ICD in CSCs.

G protein‐coupled receptors (GPCRs) can be used to shuttle peptide‐drug conjugates into cells. But, for efficient therapy, a high concentration of cargo needs to be delivered. To explore this, we studied the pharmacologically interesting neuropeptide Y1 receptor (Y1R) in one recombinant and three oncogenic cell systems that endogenously express the receptor. We demonstrate that recycled receptors behave identically to newly synthesized receptors with respect to ligand binding and internalization pathways. Depending on the cell system, biosynthesis, recycling efficiency, and peptide uptake differ partially, but shuttling was efficient in all systems. However, by comparing continuous application of the ligand for four hours to four cycles of internalization and recycling in between, a significantly higher amount of peptide uptake was achieved in the pulsed application (150–250 % to 300–400 %). Accordingly, in this well‐suited drug shuttle system pulsed application is superior under all investigated conditions and should be considered for innovative, targeted drug delivery in general., The sensitive equilibrium of receptor activation and desensitization is complex, and detailed knowledge is required for applications that use GPCRs as a shuttle system for intracellular drug delivery. As Y1R is overexpressed in various cancer subtypes, its shuttling capacity was quantitatively examined in diverse cell systems and by different methods to exploit its natural ligand‐induced endocytosis for targeted therapy.

We have discovered the sirtuin‐rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+‐dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so‐called proteolysis‐targeting chimera (PROTAC) enabled small‐molecule‐induced degradation of Sirt2. Herein, we report the structure‐based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo‐tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can also be harnessed for small‐molecule‐induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be used as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular., It's degrading: We report the structure‐based development of a chloroalkylated sirtuin rearranging ligand (SirReal) that induces Sirt2 degradation mediated by Halo‐tagged E3 ubiquitin ligases. Using this approach, we show that besides cereblon, other E3 ligases such as parkin, can be harnessed for small‐molecule‐induced Sirt2 degradation.

Hydration is a critical factor in the ligand binding process. Herein, to examine the hydration states of ligand binding sites, the three‐dimensional distribution function for the water oxygen site, gO(r), is computed for 3,706 ligand‐free protein structures based on the corresponding small molecule–protein complexes using the 3D‐RISM theory. For crystallographic waters (CWs) close to the ligand, gO(r) reveals that several CWs are stabilized by interaction networks formed between the ligand, CW, and protein. Based on the gO(r) for the crystallographic binding pose of the ligand, hydrogen bond interactions are dominant in the highly hydrated regions while weak interactions such as CH‐O are dominant in the moderately hydrated regions. The polar heteroatoms of the ligand occupy the highly hydrated and moderately hydrated regions in the crystallographic (correct) and wrongly docked (incorrect) poses, respectively. Thus, the gO(r) of polar heteroatoms may be used to distinguish the correct binding poses., The hydration states of ligand binding sites are comprehensively analyzed using a theory of solvation. An analysis of the hydration states at the positions of ligand heavy atoms indicates that the polar heteroatoms of the ligand tend to occupy highly hydrated regions in the correct ligand poses and moderately hydrated regions in the incorrect ligand poses, suggesting a way to distinguish these two poses.

Cyclic RGD peptides are well‐known ligands of integrins. The integrins αVβ3 and α5β1 are involved in angiogenesis, and integrin αVβ3 is abundantly present on cancer cells, thus representing a therapeutic target. Hence, synthetic and biophysical studies continuously are being directed towards the understanding of ligand‐integrin interaction. In this context, the development of versatile synthetic strategies to obtain fluorescent building blocks that can add molecular diversity and modular spectral characteristics while not compromising binding affinity or selectivity is a relevant task. An on‐resin intramolecular Suzuki–Miyaura cross‐coupling (SMC) between l‐ or d‐7‐bromotryptophan (7BrTrp) and a phenothiazine (Ptz) boronic acid affords fluorescent cyclic RGD pseudopeptides, c(RGD(W/w)Ptz). Ring closure by SMC establishes a phenothiazine–indole moiety with axial chirality. An array of eight novel compounds has been synthesized, among them one fluorescent compound with good affinity to integrin αVβ3. The fluorescence properties of the analogues can be efficiently tuned depending on the substituents in Ptz moiety even for fluorescence emission in the visible (red) spectral range., Shiny RGD peptides: Cyclic peptides containing the tripeptide sequence Arg‐Gly‐Asp (RGD) have been equipped with a phenothiazine‐indole biaryl moiety by Suzuki–Miyaura cross‐coupling. Modulation of the spectroscopic properties by substituents at the phenothiazine provides blue and red fluorescent RGD derivatives that selectively bind to integrin αVβ3 in the best case with nanomolar affinity.